Binding molecules specific for her3 and uses thereof

ABSTRACT

The present invention relates to antibodies and antigen binding fragments thereof that bind the extracellular domain of the HER3 receptor and inhibit various HER3 receptor related functions via ligand-dependent and/or ligand-independent mechanisms. Also provided are compositions with increased half-life. In addition, the invention provides compositions and methods for diagnosing and treating diseases associated with HER3 mediated signal transduction.

This application is a divisional of U.S. patent application Ser. No.14/359,864, which is the National Stage of International ApplicationPCT/US2012/066038, filed Nov. 20, 2012, which claims the benefit of U.S.Provisional Application No. 61/563,092, filed Nov. 23, 2011, U.S.Provisional Application No. 61/656,670, filed Jun. 7, 2012, and U.S.Provisional Application No. 61/722,558, filed Nov. 5, 2012, each ofwhich is incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application incorporates by reference a Sequence Listing submittedwith this application as text file entitled “Her3-100WO1_SL” created onNov. 12, 2012 and having a size of 31.3 kilobytes.

FIELD OF THE INVENTION

The present invention provides compositions that specifically bind toHER3 and methods for the use of such compositions for the treatment ofcancer.

BACKGROUND ART

The human epidermal growth factor receptor 3 (HER3, also known as Erbb3)is a receptor protein tyrosine and belongs to the epidermal growthfactor receptor (EGFR) EGFR/HER subfamily of receptor protein tyrosinekinases (RTK), consisting of EGFR (HER1/Erbb1), HER2/Erbb2, HER3/Erbb3and HER4/Erbb4. EGFR and HER2 are among the most well-establishedoncogenic RTKs driving the tumorigenesis of multiple types of solidtumors, including major categories such as breast, colorectal, and lungcancers. The tyrosine kinase activities of EGFR and HER2 have been shownto be essential for their oncogenic activities.

Like the prototypical EGFR, the transmembrane receptor HER3 consists ofan extracellular ligand-binding domain (ECD), a dimerization domainwithin the ECD, an transmembrane domain, and intracellular proteintyrosine kinase domain (TKD) and a C-terminal phosphorylation domain(see, e.g., Kim et al. (1998), Biochem. J. 334, 189-195; Roepstorff etal. (2008) Histochem. Cell Biol. 129, 563-578).

The ligand Heregulin (HRG) binds to the extracellular domain of HER3 andactivates the receptor-mediated signaling pathway by promotingdimerization with other EGFR family members (e.g., other HER receptors)and transphosphorylation of its intracellular domain. HER3 has beenshown to lack detectable tyrosine kinase activity, likely due to anon-conservative replacement of certain key residues in the tyrosinekinase domain. Therefore, a consequence of this kinase-deficiency, HER3needs to form hetero-dimers with other RTKs, especially EGFR and HER2,to undergo phosphorylation and be functionally active.

The central role for HER3 in oncogenesis is acting as a scaffoldingprotein to enable the maximum induction of the PI3K/AKT pathway. HER3has been shown to contain a cluster of six C-terminaltyrosine-containing motifs that when phosphorylated, mimics theconsensus PI3K/p85 binding site. Hence by forming heterodimers withHER3, the upstream onco-drivers, EGFR, HER2, cMET and FGFR2, can couplemost efficiently to the PI3K/AKT pathway. Therefore, it is reasonable toexpect that a loss of HER3 activity can block cancer progression indiverse systems driven by divergent RTKs. Studies have shown that HER3siRNA knockdown in HER2-amplified breast cancer cells led to similaranti-proliferation effects as HER2 siRNA knockdown, furtherdemonstrating the cancer's critical need for HER3.

Besides promoting tumor growth in unstressed conditions, HER3 has beenfound to be highly involved in conferring therapeutic resistances tomany targeted drugs, including EGFR tyrosine kinase inhibitors, HER2monoclonal antibodies such as trastuzumab, as well as small moleculeinhibitors of PI3K or AKT or MEK. This adds another layer of attractionto HER3 as a promising cancer target for both primary tumor debulking aswell as combating cancer resistance issues that invariably come updespite initial clinical responses.

HER3 has two different ways to dimerize with its partner RTKs:ligand-dependent (in the presence of HRG) or ligand-independent. Interms of HER2-HER3 dimers, it is known that in cells with low to mediumHER2 expression, HER3 can only complex with HER2 after ligand-binding;in contrast, in cells with amplified HER2 (HER2 IHC 3+), they formspontaneous dimers without HRG (Junttila et al. (2009) Cancer Cell.15(5):429-40). The dimers formed in the presence or absence of theligand are structurally distinct as was demonstrated by an earlier studyshowing that trastuzumab/Herceptin® (Genentech/Roche HER2 monoclonalantibody approved for HER2 3+ breast cancers) can only disrupt theligand-independent dimer but not the ligand-dependent dimer, whereaspertuzumab\Omnitarg® (rhuMAb 2C4, Genentech/Roche HER2 monoclonalantibody in phase 3 trials) can only disrupt the ligand-dependentdimers.

Dimer formation between HER family members expands the signalingpotential of HER3 and is a means not only for signal diversification butalso for signal amplification. HER3 has been shown to be phosphorylatedin a variety of cellular contexts. For example, HER3 is constitutivelyphosphorylated on tyrosine residues in a subset of human breast cancercells overexpressing HER3 (see, e.g., Kraus et al. (1993) Proc. Natl.Acad. Sci. USA 90, 2900-2904; Kim et al. (1998), Biochem. J. 334,189-195; Schaefer et al. (2004) Cancer Res. 64, 3395-3405; Schaefer etal. (2006) Neoplasia 8, 612-622). Accordingly, therapies thateffectively interfere with HER3 phosphorylation are desirable.

In addition, HER3 has been found to be overexpressed and/oroveractivated in several types of cancers such as breast cancer, ovariancancer, prostate cancer, liver cancer, kidney and urinary bladdercancers, pancreatic cancers, brain cancers, hematopoietic neoplasms,retinoblastomas, melanomas, colorectal cancers, gastric cancers, headand neck cancers, lung cancer, etc. (see, e.g., Sithanandam & Anderson(2008) Cancer Gene Ther. 15, 413-448). In general, HER3 is frequentlyactivated in EGFR, HER2, C-Met, and FGFRII-expressing cancers.

A correlation between the expression of HER2/HER3 and the progressionfrom a non-invasive to an invasive stage has been shown (Alimandi etal., Oncogene 10, 1813-1821; DeFazio et al., Cancer 87, 487-498; Naiduet al., Br. J. Cancer 78, 1385-1390). Thus, HER3 can be used as adiagnostic marker for increased tumor aggressiveness and poor survival.Sustained HER3 activation of PI3K/AKT has been repetitively shown toaccount for tumor resistance to EGFR/HER2 inhibitors.

Although the role of HER3 in the development and progression of cancerhas been explored (see, e.g., Horst et al. (2005) Int. J. Cancer 115,519-527; Xue et al. (2006) Cancer Res. 66, 1418-1426), HER3 remainslargely unappreciated as a target for clinical intervention. Mostcurrent immunotherapies primarily focus on inhibiting the action of HER2and, in particular, heterodimerization of HER2/HER3 complexes (see,e.g., Sliwkowski et al. (1994) J. Biol. Chem. 269, 14661-14665). Thus,it is an object of the present invention to provide improvedimmunotherapeutic agents that effectively inhibit HER3-mediated cellsignaling that can be used for diagnosis, prognosis prediction, andtreatment of a variety of cancers.

BRIEF SUMMARY OF THE INVENTION

The disclosure provides anti-HER3 binding molecules, e.g., antibodies orantigen-binding fragments thereof, e.g., monoclonal antibodies capableof suppressing HER3 activity in both ligand-dependent and independentsettings. In contrast, other anti-HER3 monoclonal antibodies in the art(e.g., Ab #6 (International Patent Publication WO 2008/100624) and U1-59(International Patent Publication WO 2007077028; also referred to hereinas AMG), can only suppress ligand-dependent HER3 activity. Alsodisclosed are affinity matured anti-HER3-binding molecules withincreased potency and extended half-life, which consequently can beadministered less frequently, at an increased inter-dose interval, andin smaller dose volumes. The disclosure also provides methods oftreating diseases such as cancer in a human subject comprisingadministration of an anti-HER3 binding molecule. In some specificaspects a 2C2-derived YTE mutant human antibody is used.

The disclosure provides an isolated binding molecule or antigen-bindingfragment thereof which specifically binds to an epitope within theextracellular domain of HER3, wherein the binding molecule specificallybinds to the same HER3 epitope as an antibody or antigen-bindingfragment thereof comprising the heavy chain variable region (VH) andlight chain variable region (VL) of CL16 or 2C2. Also provided is anisolated binding molecule or antigen-binding fragment thereof whichspecifically binds to HER3, and competitively inhibits HER3 binding byan antibody or antigen-binding fragment thereof comprising the VH and VLof CL16 or 2C2.

The disclosure also provides an isolated binding molecule or antigenbinding fragment thereof which specifically binds to HER3 comprising anantibody VL, wherein the VL comprises the amino acid sequence:

[FW₁]X₁GSX₂SNIGLNYVS (SEQ ID NO: 49) [FW₂]RNNQRPS (SEQ ID NO: 21)[FW₃]AAWDDX₃X₄X₅GEX₆ (SEQ ID NO: 50) [FW₄]

wherein [FW₁], [FW₂], [FW₃] and [FW₄] represent VL framework regions,and

wherein

-   -   (a) X₁ represents amino acid residues Arginine (R) or Serine        (S),    -   (b) X₂ represents amino acid residues Serine (S) or Leucine (L),    -   (c) X₃ represents amino acid residues Serine (S) or Glycine (G),    -   (d) X₄ represents amino acid residues Leucine (L) or Proline        (P),    -   (e) X₅ represents amino acid residues Arginine (R), Isoleucine        (I), Proline (P) or Serine (S), and    -   (f) X₆ represents amino acid residues Valine (V) or Alanine (A).

Furthermore, the disclosure provides an isolated binding molecule orantigen binding fragment thereof which specifically binds to HER3comprising an antibody VH, wherein the VH comprises the amino acidsequence:

[FW₅]YYYMQ (SEQ ID NO: 31) [FW₆]X₇IGSSGGVTNYADSVKG (SEQ ID NO: 51)[FW₇]VGLGDAFDI (SEQ ID NO: 35) [FW₈]

wherein [FW₅], [FW₆], [FW_(D)] and [FW₈] represent VH framework regions,and

wherein X₇ represents amino acid residues Tyrosine (Y), Isoleucine (I)or Valine (V).

The disclosure provides an isolated binding molecule or antigen bindingfragment thereof which specifically binds to HER3 comprising an antibodyVL and an antibody VH, wherein the VL comprises the amino acid sequence:

-   -   [FW₁]X₁GSX₂SNIGLNYVS(SEQ ID NO:49)[FW₂]RNNQRPS(SEQ ID NO:21)        [FW₃]AAWDDX₃X₄X₅GEX₆(SEQ ID NO:50)[FW₄]

wherein [FW₁], [FW₂], [FW₃] and [FM₄] represent VL framework regions,and

wherein

-   -   (a) X₁ represents amino acid residues Arginine (R) or Serine        (S),    -   (b) X₂ represents amino acid residues Serine (S) or Leucine (L),    -   (c) X₃ represents amino acid residues Serine (S) or Glycine (G),    -   (d) X₄ represents amino acid residues Leucine (L) or Proline        (P),    -   (e) X₅ represents amino acid residues Arginine (R), Isoleucine        (I), Proline (P) or Serine (S), and    -   (f) X₆ represents amino acid residues Valine (V) or Alanine (A),        and

wherein the VH comprises the amino acid sequence:

[FW₅]YYYMQ (SEQ ID NO: 31) [FW₆]X₇IGSSGGVTNYADSVKG (SEQ ID NO: 51)[FW₇]VGLGDAFDI (SEQ ID NO: 35) [FW₈]

wherein [FW₅], [FW₆], [FW₇] and [FW₈] represent VH framework regions,and

wherein X₇ represents amino acid residues Tyrosine (Y), Isoleucine (I)or Valine (V).

The disclosure also provides an isolated binding molecule or antigenbinding fragment thereof which specifically binds to HER3 comprising anantibody VL, wherein the VL comprises a VL complementarity determiningregion-1 (VL-CDR1) amino acid sequence identical to, or identical exceptfor four, three, two or one amino acid substitutions to: SEQ ID NO: 18,SEQ ID NO: 19, or SEQ ID NO: 20. Also, the disclosure provides anisolated binding molecule or antigen binding fragment thereof whichspecifically binds to HER3 comprising an antibody VL, wherein the VLcomprises a VL complementarity determining region-2 (VL-CDR2) amino acidsequence identical to, or identical except for four, three, two or oneamino acid substitutions to SEQ ID NO: 21.

In addition, the disclosure provides an isolated binding molecule orantigen binding fragment thereof which specifically binds to HER3comprising an antibody VL, wherein the VL comprises a complementaritydetermining region-3 (VL-CDR3) amino acid sequence identical to, oridentical except for four, three, two, or one amino acid substitutionsto: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ IDNO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30.Also, the disclosure provides an isolated binding molecule or antigenbinding fragment thereof which specifically binds to HER3 comprising anantibody VH, wherein the VH comprises a complementarity determiningregion-1 (VH-CDR1) amino acid sequence identical to, or identical exceptfor four, three, two, or one amino acid substitutions to SEQ ID NO: 31.

Furthermore, the disclosure provides an isolated binding molecule orantigen binding fragment thereof which specifically binds to HER3comprising an antibody VH, wherein the VH comprises a complementaritydetermining region-2 (VH-CDR2) amino acid sequence identical to, oridentical except for four, three, two, or one amino acid substitutionsto: SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34. Also provided is anisolated binding molecule or antigen binding fragment thereof whichspecifically binds to HER3 comprising an antibody VH, wherein the VHcomprises a complementarity determining region-3 (VH-CDR3) amino acidsequence identical to, or identical except for four, three, two, or oneamino acid substitutions to SEQ ID NO: 35.

The disclosure provides an isolated binding molecule or antigen bindingfragment thereof which specifically binds to HER3 comprising an antibodyVL, wherein the VL comprises VL-CDR1, VL-CDR2, and VL-CDR3 amino acidsequences identical to, or identical except for four, three, two, or oneamino acid substitutions in one or more of the VL-CDRS to: SEQ ID NOs:18, 21 and 22, SEQ ID NOs: 18, 21, and 26, SEQ ID NOs: 18, 21, and 27 ,SEQ ID NOs: 20, 21, and 22, SEQ ID NOs: 19, 21, and 22, SEQ ID NOs: 18,21, and 25, SEQ ID NOs: 18, 21, and 28, SEQ ID NOs: 18, 21, and 29, SEQID NOs: 18, 21, and 30, SEQ ID NOs: 18, 21, and 23, SEQ ID NOs: 19, 21,and 23, SEQ ID NOs: 20, 21, and 23, SEQ ID NOs: 18, 21, and 24, or SEQID NOs: 18, 21, and 25, respectively. The disclosure also provides anisolated binding molecule or antigen binding fragment thereof whichspecifically binds to HER3 comprising an antibody VH, wherein the VHcomprises VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences identicalto, or identical except for four, three, two, or one amino acidsubstitutions in one or more of the VH-CDRS to: SEQ ID NOs: 31, 32 and35, SEQ ID NOs: 31, 33, and 35, or SEQ ID NOs: 31, 34, and 35,respectively.

In addition, the disclosure provides an isolated antibody orantigen-binding fragment thereof which specifically binds to HER3comprising a VL and a VH comprising VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1,VH-CDR2, and VH-CDR3 amino acid sequences identical or identical exceptfor four, three, two, or one amino acid substitutions in one or moreCDRs to: SEQ ID NOs: 18, 21, 22, 31, 32, and 35, SEQ ID NOs: 18, 21, 26,31, 32 and 35, SEQ ID NOs: 18, 21, 27, 31, 32 and 35, SEQ ID NOs: 20,21, 22, 31, 32 and 35, SEQ ID NOs: 19, 21, 22, 31, 32 and 35, SEQ IDNOs: 18, 21, 25, 31, 32 and 35, SEQ ID NOs: 18, 21, 28, 31, 32 and 35,SEQ ID NOs: 18, 21, 29, 31, 32 and 35, SEQ ID NOs: 18, 21, 30, 31, 32and 35, SEQ ID NOs: 18, 21, 23, 31, 32 and 35, SEQ ID NOs: 19, 21, 23,31, 32 and 35, SEQ ID NOs: 20, 21, 23, 31, 32 and 35, SEQ ID NOs: 18,21, 24, 31, 32 and 35, or SEQ ID NOs: 18, 21, 25, 31, 32 and 35,respectively. Also provided is an isolated binding molecule or antigenbinding fragment thereof which specifically binds to HER3 comprising anantibody VL and an antibody VH, wherein the VL comprises an amino acidsequence at least about 90% to about 100% identical to a reference aminoacid sequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ IDNO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. The disclosure also providesan isolated binding molecule or antigen binding fragment thereof whichspecifically binds to HER3 comprising an antibody VL and an antibody VH,wherein the VH comprises an amino acid sequence at least about 90% toabout 100% identical to a reference amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 12 and SEQ ID NO: 13.Furthermore, the disclosure provides an isolated antibody or antigenbinding fragment thereof which specifically binds to HER3, wherein theantibody or antigen binding fragment comprises a VL comprising asequence at least about 90% to about 100% identical to a reference aminoacid sequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ IDNO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and wherein the antibody orantigen binding fragment comprises a VH comprising a sequence at leastabout 90% to about 100% identical to a reference amino acid sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 12 andSEQ ID NO: 13.

The disclosure also provides an isolated antibody or antigen bindingfragment thereof, which comprises a VL comprising SEQ ID NO: 49 and a VHcomprising SEQ ID NO: 50. In addition, the disclosure provides anisolated antibody or antigen binding fragment thereof, which comprises aVL comprising SEQ ID NO: 3 and a VH comprising SEQ ID NO: 2. Further,the disclosure provides an isolated binding molecule or antigen-bindingfragment thereof which specifically binds to an epitope within theextracellular domain of HER3, comprising an antibody VL of SEQ ID NO:3,an antibody VH of SEQ ID NO: 2, and an IgG1 constant region of SEQ ID46. Also provided is an isolated binding molecule or antigen-bindingfragment thereof which specifically binds to an epitope within theextracellular domain of HER3, consisting of an antibody VL of SEQ ID NO:3, an antibody VH of SEQ ID NO: 2, and an IgG1 constant region of SEQ ID46.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows the internalization of Clone 16 anti-HER3 monoclonalantibodies in KPL4 cells shown as depletion of surface fluorescentstaining. The top panel shows internalization at time=0. The bottompanels show internalization after 2.5 hours.

FIG. 2A shows a multiple sequence alignment corresponding to the VLsequences of anti-HER3 monoclonal antibodies Clone 16 (CL16; original,parent clone; SEQ ID NO:17), Clone 16 (GL; germlined clone; SEQ IDNO:1), 5H6 (SEQ ID NO:4), 8A3 (SEQ ID NO:5), 4H6 (SEQ ID NO:6), 6E.3(SEQ ID NO:7), 2B11 (SEQ ID NO:8), 2D1 (SEQ ID NO:9), 3A6 (SEQ ID NO:10)and 4C4 (SEQ ID NO:11). The location of CDR1, CDR2, and CDR3 isindicated. Amino acid residues which differ with respect to the CL16(GL) antibody are highlighted.

FIG. 2B shows a multiple sequence alignment corresponding to the VHsequences of anti-HER3 monoclonal antibodies Clone 16 (CL16; parentclone; SEQ ID NO: 2), and clones 15D12.1 (also referred to as 15D12.I;SEQ ID NO 12) and 15D12.2 (also referred to as 15D12.V; SEQ ID NO 13).The locations of CDR1, CDR2, and CDR3 are indicated. Amino acid residueswhich differ with respect to the CL16 parent antibody are highlighted.

FIG. 2C shows a multiple sequence alignment corresponding to the VLsequences of anti-HER3 monoclonal antibodies CL16 (original, parentclone; SEQ ID NO: 17), CL16 (GL; germlined clone; SEQ ID NO: 1), 1A4(SEQ ID NO: 14), 2C2 (SEQ ID NO: 3), 3E.1 (SEQ ID NO: 15), 2F10 (SEQ IDNO: 16), and 2B11 (SEQ ID NO: 8). The location of CDR1, CDR2, and CDR3is indicated. Amino acid residues which differ with respect to the CL16(GL) antibody are highlighted.

FIG. 3 shows suppression of HER3 phosphorylation (pHER3) inligand-driven MCF-7 cells, where HER3 is only activated by exogenous HRG(ligand). The 2C2 anti-HER3 monoclonal, published anti-HER3 monoclonalantibodies AMG and MM, and R347 control antibody were assayed. Maximumpercentages of pHER3 inhibition and IC₅₀'s are presented.

FIG. 4 shows growth suppression in MDA-MB-175 cells, an establishedHRG-autocrine loop driven model wherein endogenous HRG drives HER3activity and cell growth. The 2C2 anti-HER3 monoclonal, publishedanti-HER3 monoclonal antibodies AMG and MM, and R347 control antibodywere assayed. Maximum percentages of growth inhibition and IC₅₀'s arepresented.

FIG. 5 shows growth suppression in HMCB cells, an establishedHRG-autocrine loop driven model wherein endogenous HRG drives HER3activity and cell growth. The 2C2 anti-HER3 monoclonal, publishedanti-HER3 monoclonal antibodies AMG and MM, and R347 control antibodywere assayed. IC₅₀'s are presented.

FIG. 6 shows that 2C2 not only inhibited HMCB cell growth but alsosuppressed HER3 phosphorylation (pHER3) and AKT phosphorylation (pAKT)in this ligand dependent melanoma.

FIG. 7 shows that 2C2 suppressed HER3 phosphorylation (pHER3) and AKTphosphorylation (pAKT) in the ligand dependent A549 NSCLC.

FIGS. 8A-E shows suppression of HER3 phosphorylation (pHER3) in cellmodels for Lung Gastric and Breast cancer. FIG. 8A shows suppression ofpHER3 in the HCC827 cell line, a mutant EGFR-driven NSCLC model withEGFR/HER3 cross-talk. FIG. 8B shows suppression of pHER3 in anEGFR-TKI-resistant HCC827 NSCLC model obtained through long-termtreatment with EGFR TKI. FIG. 8C shows suppression of pHER3 in the MKN45cell line, a cMET-amplified gastric cancer model with cMET-HER3cross-talk. FIG. 8D shows suppression of pHER3 in the Kato III cellline, an FGFR2-amplified gastric cancer model with FGFR2-HER3cross-talk. FIG. 8E shows suppression of pHER3 in the BT-474 cell line,a HER2-amplified breast cancer ligand-independent model (i.e., cellslack HRG expression). The 2C2 anti-HER3 monoclonal, published anti-HER3monoclonal antibodies AMG and MM, and R347 control antibody wereassayed. Maximum percentages of pHER3 inhibition and IC₅₀'s arepresented.

FIGS. 9A-C shows suppression of AKT phosphorylation (pAKT) in cellmodels for gastric and breast cancer. FIG. 9A shows suppression of pAKTin the MKN45 cell line. FIG. 9B shows suppression of pAKT in the KatoIII cell line. FIG. 9C shows suppression of pAKT in the BT-474 cellline, a HER2-amplified breast cancer ligand-independent model (i.e.,cells lack HRG expression). The 2C2 anti-HER3 monoclonal, publishedanti-HER3 monoclonal antibodies AMG and MM, and R347 control antibodywere assayed. Maximum percentages of pAKT inhibition and IC₅₀'s arepresented.

FIGS. 10A-B shows 2C2 suppresses cell signaling and proliferation inMDA-MB-361 cells. FIG. 10A shows that 2C2 suppressed HER3phosphorylation (pHER3) in HER2-amplified MDA-MB-361 cells. FIG. 10Bshows that 2C2 suppressed cell growth in a dose dependent manner. Thepercent inhibition is shown for 6 and 14 day treatments (top and bottompanels, respectively).

FIG. 11 shows that 2C2 suppressed HER3 phosphorylation (pHER3) in HARA-Bcells expressing high levels of HRG.

FIGS. 12A-B shows that 2C2 and rhuMab 2C4, but not the EGFR antagonistscetuximab or gefitinib, inhibit HRG ligand-dependent signaling (bottomof FIG. 12A and FIG. 12B). The top portion of FIG. 12A and FIG. 12B arebasal cells, SW620 (FIG. 12A, left), SW480 (FIG. 12A, middle), Colo205(FIG. 12A, right), LOVO (FIG. 12B, left), HCT15 (FIG. 12B, middle) andCaco-2 (FIG. 12B, right).

FIG. 13 shows an HRG-HER3 ELISA binding assay measuring the directblocking of HRG binding to HER3 by the Clone 16, published AMG and MManti-HER3 monoclonal antibodies, a positive control ligand-blockinganti-HER3 monoclonal antibody, and the R347 control antibody.

FIGS. 14A-B shows 2C2 blocks HER2-HER3 dimerization. FIG. 14A shows aHRG-inducible HER2-HER3 dimerization assay that assesses the extent ofHER2-HER3 complex formation in T-47D cells, a ligand-dependent modelshowing clear HRG-induced HER2-HER3 association, pre-treated with 2C2,CL16, AMG and MM anti-HER3 monoclonal antibodies. All anti-HER3antibodies blocked this ligand-induced HER2-HER3 dimerization. FIG. 14Bshows a ligand-independent HER2-HER3 dimerization assay that assessesthe extent of HER2-HER3 complex formation in BT-474 cells, pre-treatedwith 2C2 or CL16 blocked this ligand-independent HER2-HER3 dimerization.

FIGS. 15A-B shows HER3 internalization and degradation induced by 2C2.FIG. 15A shows a FACS-based internalization assay that quantifies timecourse and extent of target internalization in response to two different2C2 monoclonal antibody concentrations. FIG. 15B shows HER3 degradationin model colorectal cancer cells Lovo, HCT15, and SW620 pretreated withanti-HER3 2C2 monoclonal antibody, or the R347 control antibody.

FIG. 16 shows a FACS-based cell-cycle analysis demonstrating that inSkBR3 cells, a HER2-amplified breast cancer cell-line similar to BT-474,both Herceptin® (trastuzumab) and CL16 monoclonal antibody (parentallead for the 2C2 monoclonal antibody) caused cell-cycle arrest at theG1-phase. Results corresponding to cells treated with the R347 controlantibody and with the rhuMAb 2C4 anti-HER2 monoclonal antibody(pertuzumab/Omnitarg®) are also shown.

FIGS. 17A-B shows inhibition of HRG induced VEGF secretion by anti-HER3antibodies. FIG. 17A shows changes in VEGF secretion in BT-474 breastcancer cells pretreated with anti-HER3 monoclonal antibodies CL16 andMerrimack MM, anti-HER2 monoclonal antibody Herceptin® (trastuzumab), orthe R347 control antibody. FIG. 17B shows changes in VEGF secretion inMCF-7 model breast cancer cells pretreated with anti-HER3 monoclonalantibodies CL16 and Merrimack MM, anti-HER2 monoclonal antibodyHerceptin® (trastuzumab), or the R347 control antibody.

FIGS. 18A-B shows that the anti-HER3 monoclonal antibody 2C2 binds tocell-surface based cyno HER3 ectopically expressed in Ad293 cells andmodulates its activity. FIG. 18A shows a Western blot analysis of Ad293cells transfected with a control vector (left side) or a vectorexpressing cyno HER3 (right side). The cells were treated with 2C2 or acontrol antibody (R347) with or without co-stimulation with HRG andprobed with anti-HER3 (middle blot), anti-pHER3 (top blot), andanti-GAPDH (bottom blot) antibodies. FIG. 18B represents thedensitometry-based quantification of pHER3 in the upper four lanes ofPanel A.

FIGS. 19A-B shows a dose-dependent reduction in tumor volume afteradministration of the 2C2 monoclonal antibody using the human FADU headand neck xenograft model. FIG. 19A shows that 7 mg/kg of 2C2administered twice per week was maximally efficacious at 99% dTGI (tumorgrowth inhibition) in this model. FIG. 19B shows strong reduction intumor volume after the combined administration of the 2C2 monoclonalantibody with the anti-EGFR monoclonal antibody cetuximab using thehuman FADU head and neck xenograft model. The combination treatmentproduced 7 out of 10 partial regressions and 2/10 complete regressions.

FIG. 20 shows non-linear pharmacokinetics for 2C2 after single dose andrepeat-dose administration of 5 mg/kg or 30 mg/kg to tumor-bearing mice.Data suggest that mouse HER3 serves as a sink to bind 2C2 administeredto the mice and that 30 mg/kg as a single dose is sufficient to saturatethe sink.

FIG. 21 shows the anti-tumor benefit of a 10 mg/kg loading dose of themonoclonal antibody 2C2 using the human FADU head and neck xenograftmodel. Administration of a loading dose of 2C2 to saturate the mouseHER3 sink enabled 2C2 at 3 mg/kg to demonstrate strong anti-tumoractivity while 3 mg/kg of 2C2 without a loading dose has only modestactivity.

FIG. 22 shows that treatment with 2C2-YTE reduces the levels of pHER3and pAKT in FADU xenograft tumor extracts. In this experiment the levelsof pHER3 and pAKT were reduced by 59.5% and 51.7%, respectively. Nochange was seen in total HER3 levels in this experiment.

FIGS. 23A-B shows a dose-dependent reduction in tumor volume afteradministration of the 2C2 monoclonal antibody using the human Detroit562head and neck xenograft model. FIG. 23A shows that 10 mg/kg of 2C2administered twice per week was maximally efficacious at 72% dTGI. FIG.23B shows a reduction in tumor volume after the combined administrationof the 2C2 monoclonal antibody with the anti-EGFR monoclonal antibodycetuximab using the human Detroit562 head and neck xenograft model. Thecombination treatment produced 9 out of 10 partial regressions whilecetuximab alone produced 5/10 partial regressions. The Detroit562xenograft model contains a PIK3CA mutation.

FIG. 24 shows a dose dependent reduction in tumor volume after theadministration of the 2C2-YTE monoclonal antibody using the human CAL27head and neck xenograft model.

FIGS. 25A-B shows a dose-dependent reduction in tumor volume afteradministration of the 2C2 monoclonal antibody using the human A549 NSCLCxenograft model. FIG. 25A shows that 30 mg/kg of 2C2 administered twiceper week was maximally efficacious at 91% dTGI up to the last day of thetreatment phase (day 33; regrowth afterwards). 2C2-YTE and 2C2 both at10 mg/kg have comparable activity. FIG. 25B shows a reduction in tumorvolume after the combined administration of the 2C2 monoclonal antibodywith the anti-EGFR monoclonal antibody cetuximab using the human A549NSCLC xenograft model. The addition of cetuximab to 2C2 increased theactivity of 2C2 during the treatment phase and delayed tumor regrowthduring the tumor regrowth phase. The A549 xenograft model contains aKRAS mutation and a LKB-1 deletion.

FIG. 26 shows a reduction in tumor volume after administration of the2C2-YTE monoclonal antibody using the human HARA-B squamous cellcarcinoma xenograft model. 30 mg/kg of 2C2-YTE administered twice perweek was maximally efficacious at 64.6% dTGI. 2C2-YTE at 10 mg/kg hadcomparable activity while 2C2-YTE at 3 mg/kg was not active.

FIG. 27 shows a dose-dependent reduction in tumor volume afteradministration of the 2C2 monoclonal antibody using the human HT-29colorectal xenograft model. 30 mg/kg of 2C2 administered twice per weekwas maximally efficacious at 56% dTGI up to the last day of thetreatment phase (day 26; regrowth afterwards). 2C2-YTE and 2C2 both at30 mg/kg have comparable activity. The HT-29 xenograft model contains aBRAF mutation.

FIG. 28 shows a reduction in tumor volume after administration of the2C2 monoclonal antibody using the human HCT-116 colorectal xenograftmodel. 30 mg/kg of 2C2 administered twice per week was maximallyefficacious at 43% dTGI. 2C2-YTE and 2C2 both at 10 mg/kg havecomparable activity. The HCT-116 xenograft model contains a KRASmutation.

FIG. 29 shows a reduction in tumor volume after administration of the2C2 monoclonal antibody using the human LOVO colorectal xenograft model.30 mg/kg of 2C2 administered twice per week was maximally efficacious at48% dTGI. 2C2-YTE and 2C2 both at 10 mg/kg have comparable activity. TheLOVO xenograft model contains a KRAS mutation.

FIG. 30 shows a reduction in tumor volume after administration of the2C2 monoclonal antibody using the human DU145 prostate xenograft model.30 mg/kg of 2C2 administered twice per week was maximally efficacious at77% dTGI. The DU145 xenograft model contains a LKB-1 deletion.

FIGS. 31A-C shows a reduction in tumor volume after administration ofthe 2C2 monoclonal antibody using the human BT-474 breast cancerorthotopic xenograft model. FIG. 31A shows 30 mg/kg of 2C2 administeredtwice per week was maximally efficacious at 55% dTGI. FIG. 31B shows areduction in tumor volume after the combined administration of the 2C2monoclonal antibody with the small molecule drug lapatinib using thehuman BT-474 breast cancer orthotopic xenograft model. The addition of2C2 to lapatinib increased the activity of lapatinib during thetreatment phase and modestly delayed tumor regrowth during the tumorregrowth phase. 2C2-YTE and 2C2 both at 30 mg/kg have comparableactivity during the treatment phase as monoefficacy treatments. FIG. 31Cshows a reduction in tumor volume after the administration of the 2C2monoclonal antibody using the human BT-474 breast cancer orthotopicxenograft model. Trastuzumab alone was very active in this model andlittle enhancement was seen by the addition of 2C2 in this model. TheBT-474 xenograft model contains amplified HER2 (3+ by HercepTest).

FIG. 32 shows that treatment with Clone 16 (2C2 precursor) reduces thelevels of pHER3 and pAKT in BT-474 xenograft tumor extracts. In thisexperiment the levels were of pHER3 and pAKT were reduced by 50% and46.1%, respectively. No change was seen in total HER3 levels in thisexperiment.

FIGS. 33A-B shows a reduction in tumor volume after administration ofthe 2C2 monoclonal antibody using the human MCF-7 breast cancerorthotopic xenograft model. FIG. 33A shows 10 mg/kg of 2C2 administeredtwice per week was maximally efficacious at 34% dTGI. 2C2-YTE and 2C2both at 10 mg/kg have comparable activity. FIG. 33B shows a reduction intumor volume after the combined administration of the 2C2 monoclonalantibody with the small molecule drug paclitaxel using the human MCF-7breast cancer orthotopic xenograft model. The addition of 2C2 topaclitaxel increased the activity of paclitaxel during the treatmentphase. The MCF-7 xenograft model contains low levels of HER2 (1+ byHercepTest).

FIGS. 34A-C shows a reduction in tumor volume after administration of2C2-YTE using the human MDA-MB-361 breast cancer orthotopic xenograftmodel (FIGS. 34A-C). The addition of 2C2-YTE to the monoclonal antibodytrastuzumab increased the activity of trastuzumab during the treatmentphase and delayed tumor regrowth during the tumor regrowth phase (FIG.34A). The addition of 2C2-YTE to the monoclonal antibody rhuMAb 2C4modestly increased the activity of rhuMAb 2C4 but did not delay theregrowth of the tumors (FIG. 34B). Addition of 2C2-YTE to the smallmolecule drug lapatinib increased the activity of lapatinib but did notdelay the regrowth of the tumors (FIG. 34C).

FIG. 35 shows prolonged exposure levels of the monoclonal antibody2C2-YTE in serum of naive human FcRn SCID transgenic mice compared to2C2 and Clone 16-GL after a single dose of these antibodies at 60 mg/kg.

FIG. 36 shows HER3 protein levels increase in response to treatment withthe MEK inhibitor (MEKi) selumetinib (indicated by a star). Treatmentwith the MEKi in combination with 2C2 reduces the HER3 levels back tonormal in HT-29 cells (left), LOVO (middle) and Colo205 (right) cancermodels. The levels of pHER3 were also examined in the HT-29 and LOVOmodels and shown to respond similarly.

FIGS. 37A-C shows that the combination of 2C2-YTE and selumetinibincreases the anti-tumor efficacy of either agent alone in subcutaneouscancer xenograft models and A549 (FIG. 37A, top), HT-29 (FIG. 37B, top),LOVO (FIG. 37C, top). Western blot analysis from tumor lysates (A549,HT-29 and LOVO xenograph models) of mice treated with the combinationshowed that phospho-HER3 and phospho-ERK were completely inhibited(Panels A-C, bottom).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides molecules and antigen-binding fragmentsthereof that bind to HER3. In some aspects, such molecules areantibodies and antigen-binding fragments thereof that specifically bindto HER3. Related polynucleotides, compositions comprising the anti-HER3antibodies or antigen-binding fragments thereof, and methods of makingthe anti-HER3 antibodies and antigen-binding fragments are alsoprovided. Methods of using the novel anti-HER3 antibodies, such asmethods of treating cancer in a subject and diagnostic uses, are furtherprovided.

In order that the present invention can be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

I. Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such can vary. As used in this specification andthe appended claims, the singular forms “a”, “an” and “the” includeplural referents unless the context clearly dictates otherwise. Theterms “a” (or “an”), as well as the terms “one or more,” and “at leastone” can be used interchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term and/or” as used in a phrase such as “Aand/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisinvention.

Units, prefixes, and symbols are denoted in their Systéme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects,which can be had by reference to the specification as a whole.Accordingly, the terms defined immediately below are more fully definedby reference to the specification in its entirety.

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

Amino acids are referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, are referredto by their commonly accepted single-letter codes.

The terms “HER3” and “HER3 receptor” are used interchangeably herein,and refer to the ErbB3 protein (also referred to as HER3, ErbB3 receptorin the literature) as described in U.S. Pat. No. 5,480,968 and inPlowman et al. (1990) Proc. Natl. Acad. Sci. USA 87, 4905-4909; seealso, Kani et al. (2005) Biochemistry 44, 15842-15857, and Cho & Leahy(2002) Science 297, 1330-1333. The full-length, mature HER3 proteinsequence (without leader sequence) corresponds to the sequence shown inFIG. 4 and SEQ ID NO: 4 of U.S. Pat. No. 5,480,968 minus the 19 aminoacid leader sequence that is cleaved from the mature protein.

The terms “inhibition” and “suppression” are used interchangeably hereinand refer to any statistically significant decrease in biologicalactivity, including full blocking of the activity. For example,“inhibition” can refer to a decrease of about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or 100% in biological activity. Accordingly, when theterms “inhibition” or “suppression” are applied to describe, e.g., aneffect on ligand-mediated HER3 phosphorylation, the term refers to theability of an antibody or antigen binding fragment thereof tostatistically significantly decrease the phosphorylation of HER3 inducedby an EGF-like ligand, relative to the phosphorylation in an untreated(control) cell. The cell which expresses HER3 can be a naturallyoccurring cell or cell line (e.g., a cancer cell) or can berecombinantly produced by introducing a nucleic acid encoding HER3 intoa host cell. In one aspect, the anti-HER3 binding molecule, e.g., anantibody or antigen binding fragment thereof inhibits ligand mediatedphosphorylation of HER3 by at least 10%, or at least 20%, or at least30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%,or at least 80%, or at least 905, or about 100%, as determined, forexample, by Western blotting followed by probing with ananti-phosphotyrosine antibody or by ELISA, as described in the Examplesinfra.

The term “growth suppression” of a cell expressing HER3, as used herein,refer to the ability of anti-HER3 binding molecule, e.g., an antibody orantigen-binding fragment thereof to statistically significantly decreaseproliferation of a cell expressing HER3 relative to the proliferation inthe absence of the anti-HER3 binding molecule, e.g., an antibody orantigen-binding fragment thereof. In one aspect, the proliferation of acell expressing HER3 (e.g., a cancer cell) can be decreased by at least10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%,or at least 60%, or at least 70%, or at least 80%, or at least 90%, orabout 100% when cells are contacted with an anti-HER3 binding molecule,e.g., an antibody or antigen-binding fragment thereof of the presentinvention, relative to the proliferation measured in the absence of theanti-HER3 binding molecule, e.g., an antibody or antigen-bindingfragment thereof (control conditions). Cellular proliferation can beassayed using art recognized techniques with measure rate of celldivision, the fraction of cells within a cell population undergoing celldivision, and/or rate of cell loss from a cell population due toterminal differentiation or cell death (e.g., thymidine incorporation).

The terms “antibody” or “immunoglobulin,” as used interchangeablyherein, include whole antibodies and any antigen binding fragment orsingle chains thereof.

A typical antibody comprises at least two heavy (H) chains and two light(L) chains interconnected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as VH)and a heavy chain constant region. The heavy chain constant region iscomprised of three domains, CH1, CH2, and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as VL)and a light chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed ComplementarityDetermining Regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FW). Each VH and VL is composed ofthree CDRs and four FWs, arranged from amino-terminus tocarboxy-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3,CDR3, FW4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies can mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (Clq) of the classicalcomplement system. Exemplary antibodies of the present disclosureinclude the Clone 16 (CL16) anti-HER3 antibodies (original andgermlined), affinity optimized clones including for example, theanti-HER3 2C2 antibody, and serum half-life-optimized anti-HER3antibodies including for example the anti-HER3 2C2-YTE antibody.

The term “germlining” means that amino acids at specific positions in anantibody are mutated back to those in the germ line. E.g., the CL16“germlined” antibody is generated from the original CL16 antibody byintroducing three point mutations, Y2S, E3V and M201, into FW1 of the VLregions.

The term “antibody” means an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, or combinations of the foregoingthrough at least one antigen recognition site within the variable regionof the immunoglobulin molecule. As used herein, the term “antibody”encompasses intact polyclonal antibodies, intact monoclonal antibodies,antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments),single chain Fv (scFv) mutants, multispecific antibodies such asbispecific antibodies generated from at least two intact antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be of any the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules such as toxins, radioisotopes, etc.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds, such as HER3. Ina certain aspect blocking antibodies or antagonist antibodiessubstantially or completely inhibit the biological activity of theantigen. Desirably, the biological activity is reduced by 10%, 20%, 30%,50%, 70%, 80%, 90%, 95%, or even 100%.

The term “HER3 antibody” or “an antibody that binds to HER3” or“anti-HER3” refers to an antibody that is capable of binding HER3 withsufficient affinity such that the antibody is useful as a therapeuticagent or diagnostic reagent in targeting HER3. The extent of binding ofan anti-HER3 antibody to an unrelated, non-HER3 protein is less thanabout 10% of the binding of the antibody to HER3 as measured, e.g., by aradioimmunoassay (RIA), BIACORE™ (using recombinant HER3 as the analyteand antibody as the ligand, or vice versa), or other binding assaysknown in the art. In certain aspects, an antibody that binds to HER3 hasa dissociation constant (K_(D)) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1nM, ≦10 pM, ≦1 pM, or ≦0.1 pM.

The terms “antigen binding fragment” refers to a portion of an intactantibody and refers to the antigenic determining variable regions of anintact antibody. It is known in the art that the antigen bindingfunction of an antibody can be performed by fragments of a full-lengthantibody. Examples of antibody fragments include, but are not limited toFab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, single chainantibodies, and multispecific antibodies formed from antibody fragments.

A “monoclonal antibody” refers to a homogeneous antibody populationinvolved in the highly specific recognition and binding of a singleantigenic determinant, or epitope. This is in contrast to polyclonalantibodies that typically include different antibodies directed againstdifferent antigenic determinants. The term “monoclonal antibody”encompasses both intact and full-length monoclonal antibodies as well asantibody fragments (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv)mutants, fusion proteins comprising an antibody portion, and any othermodified immunoglobulin molecule comprising an antigen recognition site.Furthermore, “monoclonal antibody” refers to such antibodies made in anynumber of ways including, but not limited to, by hybridoma, phageselection, recombinant expression, and transgenic animals.

The term “humanized antibody” refers to an antibody derived from anon-human (e.g., murine) immunoglobulin, which has been engineered tocontain minimal non-human (e.g., murine) sequences. Typically, humanizedantibodies are human immunoglobulins in which residues from thecomplementary determining region (CDR) are replaced by residues from theCDR of a non-human species (e.g., mouse, rat, rabbit, or hamster) thathave the desired specificity, affinity, and capability (Jones et al.,1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327;Verhoeyen et al., 1988, Science, 239:1534-1536). In some instances, theFv framework region (FW) residues of a human immunoglobulin are replacedwith the corresponding residues in an antibody from a non-human speciesthat has the desired specificity, affinity, and capability.

The humanized antibody can be further modified by the substitution ofadditional residues either in the Fv framework region and/or within thereplaced non-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. Nos. 5,225,539 or5,639,641.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FW) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FW regions and, with the CDRs from the other chain,contribute to the formation of the antigen-binding site of antibodies.There are at least two techniques for determining CDRs: (1) an approachbased on cross-species sequence variability (i.e., Kabat et al.Sequences of Proteins of Immunological Interest, (5th ed., 1991,National Institutes of Health, Bethesda Md.)); and (2) an approach basedon crystallographic studies of antigen-antibody complexes (Al-lazikaniet al. (1997) J. Molec. Biol. 273:927-948)). In addition, combinationsof these two approaches are sometimes used in the art to determine CDRs.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)).

The amino acid position numbering as in Kabat, refers to the numberingsystem used for heavy chain variable domains or light chain variabledomains of the compilation of antibodies in Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence can containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FW or CDR of the variable domain. For example, a heavychain variable domain can include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavychain FW residue 82.

TABLE 1 Loop Kabat AbM Chothia L1 L24-L34 L24-L34 L24-L34 L2 L50-L56L50-L56 L50-L56 L3 L89-L97 L89-L97 L89-L97 H1 H31-H35B H26-H35B H26-H32. . . 34 (Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 (ChothiaNumbering) H2 H50-H65 H50-H58 H52-H56 H3 H95-H102 H95-H102 H95-H102

The Kabat numbering of residues can be determined for a given antibodyby alignment at regions of homology of the sequence of the antibody witha “standard” Kabat numbered sequence. Chothia refers instead to thelocation of the structural loops (Chothia and Lesk, J. Mol. Biol.196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numberedusing the Kabat numbering convention varies between H32 and H34depending on the length of the loop (this is because the Kabat numberingscheme places the insertions at H35A and H35B; if neither 35A nor 35B ispresent, the loop ends at 32; if only 35A is present, the loop ends at33; if both 35A and 35B are present, the loop ends at 34). The AbMhypervariable regions represent a compromise between the Kabat CDRs andChothia structural loops, and are used by Oxford Molecular's AbMantibody modeling software.

IMGT (ImMunoGeneTics) also provides a numbering system for theimmunoglobulin variable regions, including the CDRs. See e.g., Lefranc,M. P. et al., Dev. Comp. Immunol. 27: 55-77(2003), which is hereinincorporated by reference. The IMGT numbering system was based on analignment of more than 5,000 sequences, structural data, andcharacterization of hypervariable loops and allows for easy comparisonof the variable and CDR regions for all species. According to the IMGTnumbering schema VH-CDR1 is at positions 26 to 35, VH-CDR2 is atpositions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is atpositions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is atpositions 89 to 97.

As used throughout the specification the VH CDRs sequences describedcorrespond to the classical Kabat numbering locations, namely KabatVH-CDR1 is at positions 31-35, VH-CDR2 is a positions 50-65, and VH-CDR3is at positions 95-102. VL-CDR2 and VL-CDR3 also correspond to classicalKabat numbering locations, namely positions 50-56 and 89-97,respectively. As used herein, the terms “VL-CDR1” or “light chain CDR1”correspond to sequences located at Kabat positions 23-34 in the VL (incontrast, the classical VL-CDR1 location according to the Kabatnumbering schema corresponds to positions 24-34).

As used herein the Fc region includes the polypeptides comprising theconstant region of an antibody excluding the first constant regionimmunoglobulin domain. Thus Fc refers to the last two constant regionimmunoglobulin domains of IgA, IgD, and IgG, and the last three constantregion immunoglobulin domains of IgE and IgM, and the flexible hingeN-terminal to these domains. For IgA and IgM Fc may include the J chain.For IgG, Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2and Cγ3) and the hinge between Cgammal (Cγ1) and Cgamma2 (Cγ2). Althoughthe boundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to comprise residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index asset forth in Kabat (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). Fc may refer to this region in isolation, or thisregion in the context of an antibody, antibody fragment, or Fc fusionprotein. Polymorphisms have been observed at a number of different Fcpositions, including but not limited to positions 270, 272, 312, 315,356, and 358 as numbered by the EU index, and thus slight differencesbetween the presented sequence and sequences in the prior art may exist.

The term “human antibody” means an antibody produced by a human or anantibody having an amino acid sequence corresponding to an antibodyproduced by a human made using any technique known in the art. Thisdefinition of a human antibody includes intact or full-lengthantibodies, fragments thereof, and/or antibodies comprising at least onehuman heavy and/or light chain polypeptide such as, for example, anantibody comprising murine light chain and human heavy chainpolypeptides.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g., mouse, rat, rabbit, etc) with the desiredspecificity, affinity, and capability while the constant regions arehomologous to the sequences in antibodies derived from another (usuallyhuman) to avoid eliciting an immune response in that species.

The terms “YTE” or “YTE mutant” refer to a mutation in IgG1 Fc thatresults in an increase in the binding to human FcRn and improves theserum half-life of the antibody having the mutation. A YTE mutantcomprises a combination of three mutations, M252Y/S254T/T256E (EUnumbering Kabat et al. (1991) Sequences of Proteins of ImmunologicalInterest, U.S. Public Health Service, National Institutes of Health,Washington, D.C.), introduced into the heavy chain of an IgG1. See U.S.Pat. No. 7,658,921, which is incorporated by reference herein. The YTEmutant has been shown to increase the serum half-life of antibodiesapproximately four-times as compared to wild-type versions of the sameantibody (Dall'Acqua et al., J. Biol. Chem. 281:23514-24 (2006)). Seealso U.S. Pat. No. 7,083,784, which is hereby incorporated by referencein its entirety.

“Binding affinity” generally refers to the strength of the sum total ofnon-covalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (K_(D)). Affinity can be measured by commonmethods known in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention.

“Potency” is normally expressed as an IC₅₀ value, in nM unless otherwisestated. IC₅₀ is the median inhibitory concentration of an antibodymolecule. In functional assays, IC₅₀ is the concentration that reduces abiological response by 50% of its maximum. In ligand-binding studies,IC₅₀ is the concentration that reduces receptor binding by 50% ofmaximal specific binding level. IC₅₀ can be calculated by any number ofmeans known in the art. Improvement in potency can be determined bymeasuring, e.g., against the parent CL16 (Clone 16) monoclonal antibody.

The fold improvement in potency for the antibodies or polypeptides ofthe invention as compared to a Clone 16 antibody can be at least about2-fold, at least about 4-fold, at least about 6-fold, at least about8-fold, at least about 10-fold, at least about 20-fold, at least about30-fold, at least about 40-fold, at least about 50-fold, at least about60-fold, at least about 70-fold, at least about 80-fold, at least about90-fold, at least about 100-fold, at least about 110-fold, at leastabout 120-fold, at least about 130-fold, at least about 140-fold, atleast about 150-fold, at least about 160-fold, at least about 170-fold,or at least about 180-fold or more.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enables these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. Specific high-affinity IgGantibodies directed to the surface of target cells “arm” the cytotoxiccells and are absolutely required for such killing. Lysis of the targetcell is extracellular, requires direct cell-to-cell contact, and doesnot involve complement. It is contemplated that, in addition toantibodies, other proteins comprising Fc regions, specifically Fc fusionproteins, having the capacity to bind specifically to an antigen-bearingtarget cell will be able to effect cell-mediated cytotoxicity. Forsimplicity, the cell-mediated cytotoxicity resulting from the activityof an Fc fusion protein is also referred to herein as ADCC activity.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is a polypeptide, antibody, polynucleotide, vector,cell, or composition which is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cells orcompositions include those which have been purified to a degree thatthey are no longer in a form in which they are found in nature. In someaspects, an antibody, polynucleotide, vector, cell, or composition whichis isolated is substantially pure.

The term “subject” refers to any animal (e.g., a mammal), including, butnot limited to humans, non-human primates, rodents, and the like, whichis to be the recipient of a particular treatment. Typically, the terms“subject” and “patient” are used interchangeably herein in reference toa human subject.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the composition wouldbe administered. Such composition can be sterile.

An “effective amount” of an antibody as disclosed herein is an amountsufficient to carry out a specifically stated purpose. An “effectiveamount” can be determined empirically and in a routine manner, inrelation to the stated purpose.

The term “therapeutically effective amount” refers to an amount of anantibody or other drug effective to “treat” a disease or disorder in asubject or mammal.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label can be detectable byitself (e.g., radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, can catalyze chemical alteration of a substratecompound or composition which is detectable.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to both (1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder and (2) prophylactic or preventativemeasures that prevent and/or slow the development of a targetedpathologic condition or disorder. Thus, those in need of treatmentinclude those already with the disorder; those prone to have thedisorder; and those in whom the disorder is to be prevented. In certainaspects, a subject is successfully “treated” for cancer according to themethods of the present invention if the patient shows, e.g., total,partial, or transient remission of a certain type of cancer.

The terms “cancer”, “tumor”, “cancerous”, and “malignant” refer to ordescribe the physiological condition in mammals that is typicallycharacterized by unregulated cell growth. Examples of cancers includebut are not limited to, carcinoma including adenocarcinomas, lymphomas,blastomas, melanomas, sarcomas, and leukemias. More particular examplesof such cancers include squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, gastrointestinal cancer, Hodgkin's andnon-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma,cervical cancer, ovarian cancer, liver cancer such as hepatic carcinomaand hepatoma, bladder cancer, breast cancer (including hormonallymediated breast cancer, see, e.g., Innes et al. (2006) Br. J. Cancer94:1057-1065), colon cancer, colorectal cancer, endometrial carcinoma,myeloma (such as multiple myeloma), salivary gland carcinoma, kidneycancer such as renal cell carcinoma and Wilms' tumors, basal cellcarcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer,testicular cancer, esophageal cancer, various types of head and neckcancer and cancers of mucinous origins, such as, mucinous ovariancancer, cholangiocarcinoma (liver) and renal papillary carcinoma.

As used herein, the term “carcinomas” refers to cancers of epithelialcells, which are cells that cover the surface of the body, producehormones, and make up glands. Examples of carcinomas are cancers of theskin, lung, colon, stomach, breast, prostate and thyroid gland.

The term “KRAS mutation,” as used herein, refers to mutations found incertain cancers in a human homolog of the v-Ki-ras2 Kirsten rat sarcomaviral oncogene. Non-limiting examples of human KRAS gene mRNA sequencesinclude Genbank Accession Nos. NM004985 and NM033360. It has beenreported that KRAS mutations are found in 73% of pancreatic tumors, 35%of colorectal tumors, 16% of ovarian tumors and 17% of lung tumors. KRASmutation generally occur in codons 12 or 143 of the human KRAS gene.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase. A polynucleotidecan comprise modified nucleotides, such as methylated nucleotides andtheir analogs. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

The term “vector” means a construct, which is capable of delivering, andin some aspects, expressing, one or more gene(s) or sequence(s) ofinterest in a host cell. Examples of vectors include, but are notlimited to, viral vectors, naked DNA or RNA expression vectors, plasmid,cosmid or phage vectors, DNA or RNA expression vectors associated withcationic condensing agents, DNA or RNA expression vectors encapsulatedin liposomes, and certain eukaryotic cells, such as producer cells.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer can be linear or branched, it can comprise modifiedamino acids, and it can be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon antibodies, in certain aspects, the polypeptides can occur assingle chains or associated chains.

The terms “identical” or percent “identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity can be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software are known in the art that can be used to obtainalignments of amino acid or nucleotide sequences.

One such non-limiting example of a sequence alignment algorithm is thealgorithm described in Karlin et al., 1990, Proc. Natl. Acad. Sci.,87:2264-2268, as modified in Karlin et al., 1993, Proc. Natl. Acad.Sci., 90:5873-5877, and incorporated into the NBLAST and XBLAST programs(Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402). In certainaspects, Gapped BLAST can be used as described in Altschul et al., 1997,Nucleic Acids Res. 25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al.,1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech,South San Francisco, Calif.) or Megalign (DNASTAR) are additionalpublicly available software programs that can be used to alignsequences. In certain aspects, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (e.g., using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). Incertain alternative aspects, the GAP program in the GCG softwarepackage, which incorporates the algorithm of Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) can be used to determine the percentidentity between two amino acid sequences (e.g., using either a BLOSUM62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certainaspects, the percent identity between nucleotide or amino acid sequencesis determined using the algorithm of Myers and Miller (CABIOS, 4:11-17(1989)). For example, the percent identity can be determined using theALIGN program (version 2.0) and using a PAM120 with residue table, a gaplength penalty of 12 and a gap penalty of 4. Appropriate parameters formaximal alignment by particular alignment software can be determined byone skilled in the art. In certain aspects, the default parameters ofthe alignment software are used.

In certain aspects, the percentage identity “X” of a first amino acidsequence to a second sequence amino acid is calculated as 100×(Y/Z),where Y is the number of amino acid residues scored as identical matchesin the alignment of the first and second sequences (as aligned by visualinspection or a particular sequence alignment program) and Z is thetotal number of residues in the second sequence. If the length of afirst sequence is longer than the second sequence, the percent identityof the first sequence to the second sequence will be higher than thepercent identity of the second sequence to the first sequence.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., asparagine, glutamine, serine,threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. In certainaspects, conservative substitutions in the sequences of the polypeptidesand antibodies of the invention do not abrogate the binding of thepolypeptide or antibody containing the amino acid sequence, to theantigen(s), i.e., the HER3 to which the polypeptide or antibody binds.Methods of identifying nucleotide and amino acid conservativesubstitutions which do not eliminate antigen binding are well-known inthe art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993);Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al.,Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

The term “consensus sequence,” as used herein with respect to lightchain (VL) and heavy chain (VH) variable regions, refers to a compositeor genericized VL or VH sequence defined based on information as towhich amino acid residues within the VL or VH chain are amenable tomodification without detriment to antigen binding. Thus, in a “consensussequence” for a VL or VH chain, certain amino acid positions areoccupied by one of multiple possible amino acid residues at thatposition. For example, if an arginine (R) or a serine (S) occur at aparticular position, then that particular position within the consensussequence can be either arginine or serine (R or S). Consensus sequencesfor VH and VL chain can be defined, for example, by in vitro affinitymaturation (e.g., randomizing every amino acid position in a certain CDRusing degenerate coding primers), by scanning mutagenesis (e.g., alaninescanning mutagenesis) of amino acid residues within the antibody CDRs,or any other methods known in the art, followed by evaluation of thebinding of the mutants to the antigen to determine whether the mutatedamino acid position affects antigen binding. In some aspects, mutationsare introduced in the CDR regions. In other aspects, mutations areintroduced in framework regions. In some other aspects, mutations areintroduced in CDR and framework regions.

II. Anti-HER3-Binding Molecules

The present invention provides HER3 binding molecules, e.g., antibodiesand antigen-binding fragments thereof that specifically bind HER3. Thefull-length amino acid (aa) and nucleotide (nt) sequences for HER3 areknown in the art (see, e.g., UniProt Acc. No. P2186 for human HER3, orUniProt Acc. No. 088458 for mouse HER3). In some aspects, the anti-HER3binding molecules are human antibodies. In certain aspects, the HER3binding molecules are antibodies or antigen-binding fragments thereof.In some aspects, HER3 binding molecules, e.g., antibodies orantigen-binding fragments thereof comprise a Fab, a Fab′, a F(ab′)₂, aFd, a single chain Fv or scFv, a disulfide linked Fv, a V-NAR domain, anIgNar, an intrabody, an IgGΔCH2, a minibody, a F(ab′)₃, a tetrabody, atriabody, a diabody, a single-domain antibody, DVD-Ig, Fcab, mAb², a(scFv)₂, or a scFv-Fc. In some aspects, the antibody is of the IgG1subtype and comprises the triple mutant YTE, as disclosed supra in theDefinitions section.

In certain aspects, anti-HER3 antibodies or antigen-binding fragmentsthereof of the invention are modified compared to the parent Clone 16(CL16) antibody. The modifications can include mutations in the CDRregions and/or in the FW regions as compared to CL16. In certainaspects, an anti-HER3 antibody of the invention comprises modificationsto CDR1 and/or CDR3 of the light chain of CL16, including, but notlimited to:

1) a light chain CDR1 comprising the consensus sequenceX₁GSX₂SNIGLNYVS(SEQ ID NO:49), wherein X₁ is selected from R or S, andX₂ is selected from S or L; and

2) a light chain CDR3 comprising the consensus sequenceAAWDDX₃X₄X₅GEX₆(SEQ ID NO:50), wherein X₃ is selected from S or G, X₄ isselected from L or P, X₅ is selected from R, I, P or S, and X₆ isselected from V or A.

In certain aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises modifications to CDR2 of the heavychain of CL16, including, but not limited to a heavy chain CDR1comprising the consensus sequence X₇IGSSGGVTNYADSVKG(SEQ ID NO:51),wherein X₇ is selected from Y, I or V.

In one aspect, an anti-HER3 antibody or antigen binding fragment thereofcomprises a VL region comprising the consensus amino acid sequence:

[FW₁]X₁GSX₂SNIGLNYVS (SEQ ID NO: 49) [FW₂]RNNQRPS (SEQ ID NO: 21)[FW₃]AAWDDX₃X₄X₅GEX₆ (SEQ ID NO: 50) [FW₄]

-   -   wherein [FW₁], [FW₂], [FW₃] and [FW₄] represent the amino acid        residues of VL framework region 1 (SEQ ID NO: 40 or 44), VL        framework region 2 (SEQ ID NO: 41), VL framework region 3 (SEQ        ID NO: 42) and VL framework region 4 (SEQ ID NO: 43), and        wherein X₁ represents amino acid residues arginine (R) or serine        (S), X₂ represents amino acid residues serine (S) or leucine        (L), X₃ represents amino acid residues serine (S) or glutamic        acid (E), X₄ represents amino acid residues leucine (L) or        proline (P), X₅ represents amino acid residues arginine (R),        isoleucine (I), proline (P) or serine (5), and X₆ represents        amino acid residues valine (V) or arginine (R).

In one aspect, an anti-HER3 antibody or antigen binding fragment thereofcomprises a VH region comprises the consensus amino acid sequence:

[FW₅]YYYMQ (SEQ ID NO: 31) [FW₆]X₇IGSSGGVTNYADSVKG (SEQ ID NO: 51)[FW₇]VGLGDAFDI (SEQ ID NO: 35) [FW₈]

-   -   wherein [FW₅], [FW₆], [FW₇] and [FW₈] represent the amino acid        residues of VH framework region 1 (SEQ ID NO: 36), VH framework        region 2 (SEQ ID NO: 37), VH framework region 3 (SEQ ID NO: 38)        and VH framework region 4 (SEQ ID NO: 39), and wherein X₇        represents amino acid residues tyrosine (Y), isoleucine (I) or        valine (V).

In one aspect, an anti-HER3 antibody or antigen binding fragment thereofcomprises a VL region comprising the consensus amino acid sequence:

[FW₁]X₁GSX₂SNIGLNYVS (SEQ ID NO: 49) [FW₂]RNNQRPS (SEQ ID NO: 21)[FW₃]AAWDDX₃X₄X₅GEX₆ (SEQ ID NO: 50) [FW₄]

-   -   wherein [FW₁], [FW₂], [FW₃] and [FW₄] represent the amino acid        residues of VL framework region 1 (SEQ ID NO: 40 or 44), VL        framework region 2 (SEQ ID NO: 41), VL framework region 3 (SEQ        ID NO: 42) and VL framework region 4 (SEQ ID NO: 43), and        wherein X₁ represents amino acid residues arginine (R) or serine        (S), X₂ represents amino acid residues serine (S) or leucine        (L), X₃ represents amino acid residues serine (S) or glutamic        acid (E), X₄ represents amino acid residues leucine (L) or        proline (P), X₅ represents amino acid residues arginine (R),        isoleucine (I), proline (P) or serine (5), and X₆ represents        amino acid residues valine (V) or arginine (R); and wherein said        anti-HER3 antibody or antigen binding fragment thereof further        comprises a VH region which comprises the consensus amino acid        sequence:

[FW₅]YYYMQ (SEQ ID NO: 31) [FW₆]X₇IGSSGGVTNYADSVKG (SEQ ID NO: 51)[FW₇]VGLGDAFDI (SEQ ID NO: 35) [FW₈]

-   -   wherein [FW₅], [FW₆], [FW₇] and [FW₈] represent the amino acid        residues of VH framework region 1 (SEQ ID NO: 36), VH framework        region 2 (SEQ ID NO: 37), VH framework region 3 (SEQ ID NO: 38)        and VH framework region 4 (SEQ ID NO: 39), and wherein X₇        represents amino acid residues tyrosine (Y), isoleucine (I) or        valine (V).

In some aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises a VL-CDR1 consisting of sequenceselected from the group consisting of SEQ ID NOs: 18, 19 and 20. In someaspects, an anti-HER3 antibody or antigen-binding fragment thereof ofthe invention comprises a VL-CDR1 comprising a sequence selected fromthe group consisting of SEQ ID NOs: 18, 19 and 20. In some aspects, ananti-HER3 antibody or antigen-binding fragment thereof of the inventioncomprises a VL-CDR2 consisting of SEQ ID NO: 21. In some aspects, ananti-HER3 antibody or antigen-binding fragment thereof of the inventioncomprises a VL-CDR2 comprising SEQ ID NO: 21. In some aspects, ananti-HER3 antibody or antigen-binding fragment thereof of the inventioncomprises a VL-CDR3 consisting of a sequence selected from the groupconsisting of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, and 30. Insome aspects, an anti-HER3 antibody or antigen-binding fragment thereofof the invention comprises a VL-CDR3 comprising a sequence selected fromthe group consisting of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, and30.

In some aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises a VH-CDR1 consisting of SEQ ID NO:31. In some aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises a VH-CDR1 comprising SEQ ID NO: 31.In some aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises a VH-CDR2 consisting of a sequenceselected from the group consisting of SEQ ID NOs: 32, 33 and 34. In someaspects, an anti-HER3 antibody or antigen-binding fragment thereof ofthe invention comprises a VH-CDR2 comprising a sequence selected fromthe group consisting of SEQ ID NOs: 32, 33 and 34. In some aspects, ananti-HER3 antibody or antigen-binding fragment thereof of the inventioncomprises a VH-CDR3 consisting of SEQ ID NO: 35. In some aspects, ananti-HER3 antibody or antigen-binding fragment thereof of the inventioncomprises a VH-CDR3 comprising SEQ ID NO: 35.

In some aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises a VL-CDR1 consisting of a sequenceselected from the group consisting of SEQ ID NOs: 18, 19 and 20, exceptfor one, two, three or four amino acid substitutions. In some aspects,an anti-HER3 antibody or antigen-binding fragment thereof of theinvention comprises a VL-CDR1 comprising a sequence selected from thegroup consisting of SEQ ID NOs: 18, 19 and 20, except for one, two,three or four amino acid substitutions. In some aspects, an anti-HER3antibody or antigen-binding fragment thereof of the invention comprisesa VL-CDR2 consisting of SEQ ID NO: 21, except for one, two, three orfour amino acid substitutions. In some aspects, an anti-HER3 antibody orantigen-binding fragment thereof of the invention comprises a VL-CDR2comprising SEQ ID NO: 21, except for one, two, three or four amino acidsubstitutions. In some aspects, an anti-HER3 antibody or antigen-bindingfragment thereof of the invention comprises a VL-CDR3 consisting of asequence selected from the group consisting of SEQ ID NOs: 22, 23, 24,25, 26, 27, 28, 29, and 30, except for one, two, three or four aminoacid substitutions. In some aspects, an anti-HER3 antibody orantigen-binding fragment thereof of the invention comprises a VL-CDR3comprising a sequence selected from the group consisting of SEQ ID NOs:22, 23, 24, 25, 26, 27, 28, 29, and 30, except for one, two, three orfour amino acid substitutions.

In some aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises a VH-CDR1 consisting of SEQ ID NO:31, except for one, two, three or four amino acid substitutions. In someaspects, an anti-HER3 antibody or antigen-binding fragment thereof ofthe invention comprises a VH-CDR1 comprising SEQ ID NO: 31, except forone, two, three or four amino acid substitutions. In some aspects, ananti-HER3 antibody or antigen-binding fragment thereof of the inventioncomprises a VH-CDR2 consisting of a sequence selected from the groupconsisting of SEQ ID NOs: 32, 33 and 34, except for one, two, three orfour amino acid substitutions. In some aspects, an anti-HER3 antibody orantigen-binding fragment thereof of the invention comprises a VH-CDR2comprising a sequence selected from the group consisting of SEQ ID NOs:32, 33 and 34, except for one, two, three or four amino acidsubstitutions. In some aspects, an anti-HER3 antibody or antigen-bindingfragment thereof of the invention comprises a VH-CDR3 consisting of SEQID NO: 35, except for one, two, three or four amino acid substitutions.In some aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises a VH-CDR3 comprising SEQ ID NO: 35,except for one, two, three or four amino acid substitutions.

In some aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises a VL-CDR1 consisting of a sequenceselected from the group consisting of SEQ ID NOs: 18, 19 and 20; aVL-CDR2 consisting of SEQ ID NO: 21; and a VL-CDR3 consisting of asequence selected from the group consisting of SEQ ID NOs: 22, 23, 24,25, 26, 27, 28, 29, and 30. In some aspects, an anti-HER3 antibody orantigen-binding fragment thereof of the invention comprises a VL-CDR1comprising a sequence selected from the group consisting of SEQ ID NOs:18, 19 and 20; a VL-CDR2 comprising SEQ ID NO: 21; and a VL-CDR3comprising a sequence selected from the group consisting of SEQ ID NOs:22, 23, 24, 25, 26, 27, 28, 29, and 30.

In some aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises a VH-CDR1 consisting of SEQ ID NO:31; a VH-CDR2 consisting of a sequence selected from the groupconsisting of SEQ ID NOs: 32, 33 and 34; and a VH-CDR3 consisting of SEQID NO: 35. In some aspects, an anti-HER3 antibody or antigen-bindingfragment thereof of the invention comprises a VH-CDR1 comprising SEQ IDNO: 31; a VH-CDR2 comprising a sequence selected from the groupconsisting of SEQ ID NOs: 32, 33 and 34; a VH-CDR3 comprising SEQ ID NO:35.

In some aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises a VL-CDR1 consisting of a sequenceselected from the group consisting of SEQ ID NOs: 18, 19 and 20, exceptfor one, two, three or four amino acid substitutions; a VL-CDR2consisting of SEQ ID NO: 21, except for one, two, three or four aminoacid substitutions; and a VL-CDR3 consisting of a sequence selected fromthe group consisting of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, and30, except for one, two, three or four amino acid substitutions. In someaspects, an anti-HER3 antibody or antigen-binding fragment thereof ofthe invention comprises a VL-CDR1 comprising a sequence selected fromthe group consisting of SEQ ID NOs: 18, 19 and 20, except for one, two,three or four amino acid substitutions; a VL-CDR2 comprising SEQ ID NO:21, except for one, two, three or four amino acid substitutions; and aVL-CDR3 comprising a sequence selected from the group consisting of SEQID NOs: 22, 23, 24, 25, 26, 27, 28, 29, and 30, except for one, two,three or four amino acid substitutions.

In some aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises a VH-CDR1 consisting of SEQ ID NO:31, except for one, two, three or four amino acid substitutions; aVH-CDR2 consisting of a sequence selected from the group consisting ofSEQ ID NOs: 32, 33 and 34, except for one, two, three or four amino acidsubstitutions; and a VH-CDR3 consisting of SEQ ID NO: 35, except forone, two, three or four amino acid substitutions. In some aspects, ananti-HER3 antibody or antigen-binding fragment thereof antibody of theinvention comprises a VH-CDR1 comprising SEQ ID NO: 31, except for one,two, three or four amino acid substitutions; a VH-CDR2 comprising asequence selected from the group consisting of SEQ ID NOs: 32, 33 and34, except for one, two, three or four amino acid substitutions; andVH-CDR3 comprising SEQ ID NO: 35, except for one, two, three or fouramino acid substitutions.

In certain aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises modifications to CDR1, CDR2, and/orCDR3 of the heavy and/or light chain, and further comprisesmodifications to FW1, FW2, FW3, and/or FW4 of the heavy and/or lightchain. In some aspects, FW₁ comprises SEQ ID NO: 40 or 44, FW₂ comprisesSEQ ID NO: 41, FW₃ comprises SEQ ID NO: 42, FW₄ comprises SEQ ID NO: 43,FW₅ comprises SEQ ID NO: 36, FW₆ comprises SEQ ID NO: 37, FW₇ comprisesSEQ ID NO: 38, and FW₈ comprises SEQ ID NO: 39.

In some aspects, FW₁ comprises SEQ ID NO: 40 or 44, except for one, two,three or four amino acid substitutions; FW₂ comprises SEQ ID NO: 41,except for one, two, three or four amino acid substitutions; FW₃comprises SEQ ID NO: 42, except for one, two, three or four amino acidsubstitutions; FW₄ comprises SEQ ID NO: 43, except for one, two, threeor four amino acid substitutions; FW₅ comprises SEQ ID NO: 36, exceptfor one, two, three or four amino acid substitutions; FW₆ comprises SEQID NO: 37, except for one, two, three or four amino acid substitutions;FW₇ comprises SEQ ID NO: 38, except for one, two, three or four aminoacid substitutions; and FW₈ comprises SEQ ID NO: 39, except for one,two, three or four amino acid substitutions.

In certain aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises a VL and a VH comprising VL-CDR1,VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequencesidentical or identical except for four, three, two, or one amino acidsubstitutions in one or more CDRs to: SEQ ID NOs: 18, 21, 22, 31, 32,and 35, SEQ ID NOs: 18, 21, 26, 31, 32 and 35, SEQ ID NOs: 18, 21, 27,31, 32 and 35, SEQ ID NOs: 20, 21, 22, 31, 32 and 35, SEQ ID NOs: 19,21, 22, 31, 32 and 35, SEQ ID NOs: 18, 21, 25, 31, 32 and 35, SEQ IDNOs: 18, 21, 28, 31, 32 and 35, SEQ ID NOs: 18, 21, 29, 31, 32 and 35,SEQ ID NOs: 18, 21, 30, 31, 32 and 35, SEQ ID NOs: 18, 21, 23, 31, 32and 35, SEQ ID NOs: 19, 21, 23, 31, 32 and 35, SEQ ID NOs: 20, 21, 23,31, 32 and 35, SEQ ID NOs: 18, 21, 24, 31, 32 and 35, or SEQ ID NOs: 18,21, 25, 31, 32 and 35, respectively.

Heavy and light chain variable domains of the anti-HER3 antibody orantigen-binding fragment thereof of the invention include the sequenceslisted in TABLE 2.

TABLE 2 SEQ ID NO. Description Sequence  1 CL16 VLQSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRP (Germlined)SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGEVFGGGTKLTVL 17 CL16 VLQYELTQPPSASGTPGQRVTMSCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQR (original)PSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGEVFGGGTKLTVL  2 CL16 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSYYYMQWVRQAPGKGLEWVSYIGSSGGVTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGLGDAFD IWGQGTMVTVSS  45H6 VL QSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDGLPGEVFGGGTKLTVL  5 8A3 VLQSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLIGEVFGGGTKLTVL  6 4H6 VLQSVLTQPPSASGTPGQRVTISCRGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGEVFGGGTKLTVL  7 6E.3 VLQSVLTQPPSASGTPGQRVTISCSGSLSNIGLNYVSWYQQLPGTAPKLLISRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGEVFGGGTKLTVL  8 2B11 VLQSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLPGEVFGGGTKLTVL  9 2D1 VLQSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGEAFGGGTKLTVL 10 3A6 VLQSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSPSGEVFGGGTKLTVL 11 4C4 VLQSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLRGEVFGGGTKLTVL 12 15D12.1EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYYMQWVRQAPGKGLEWVSIIGSS (15D12.1) VHGGVTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGLGDAFDI WGQGTMVTVSS 1315D12.2 EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYYMQWVRQAPGKGLEWVSVIGS (15D12.V)SGGVTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGLGDAFD VH IWGQGTMVTVSS 141A4 VL QSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSPPGEAFGGGTKLTVL  3 2C2 VLQSVLTQPPSASGTPGQRVTISCSGSLSNIGLNYVSWYQQLPGTAPKLLISRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSPPGEAFGGGTKLTVL 15 3E.1 VLQSVLTQPPSASGTPGQRVTISCRGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSPPGEAFGGGTKLTVL 16 2F10QSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSPSGEAFGGGTKLTVL

In certain aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises an antibody VL and an antibody VH,wherein the VL comprises an amino acid sequence at least about 80%,about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about99%, or about 100% identical to a reference amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, and SEQ ID NO: 17.

In other aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises an antibody VL and an antibody VH,wherein the VH comprises an amino acid sequence at least about 80%,about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about99%, or about 100% identical to a reference amino acid sequence selectedfrom the group consisting of SEQ ID NO: 2, SEQ ID NO: 12 and SEQ ID NO:13.

In other aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises a VL comprising a sequence at leastabout 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about98%, about 99%, or about 100% identical to a reference amino acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, and SEQ ID NO: 17, and further comprises a VHcomprising a sequence at least about 80%, about 85%, about 90%, about95%, about 96%, about 97%, about 98%, about 99%, or about 100% identicalto a reference amino acid sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 12 and SEQ ID NO: 13.

In some aspects, an anti-HER3 antibody or antigen-binding fragmentthereof comprises a VH of TABLE 2 and a VL of TABLE 2. Antibodies aredesignated throughout the specification according to their VL chains.The heavy chains of the specific antibodies disclosed in the presentspecification correspond to the CL16 original heavy chain (SEQ ID NO:2). Thus, the “CL16 antibody” is an IgG1 comprising two original CL16light chains (SEQ ID NO: 17) and two CL16 original heavy chains (SEQ IDNO: 2), whereas the “2C2 antibody” is an IgG1 comprising two 2C2 lightchains (2C2 VL (SEQ ID NO: 3) and two CL16 original heavy chains (SEQ IDNO: 2).

In some aspects, the anti-HER3 antibody or antigen-binding fragmentthereof comprises a heavy chain constant region or fragment thereof. Insome specific aspects, the heavy chain constant region is an IgGconstant region. The IgG constant region can comprise a light chainconstant region selected from the group consisting of a kappa constantregion and a lambda constant region.

In certain aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention binds HER3 with substantially the same orbetter affinity as the CL16 antibody, comprising the CL16 original heavychain (SEQ ID NO: 2) and the original CL16 light chain (SEQ ID NO: 17).In certain aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention binds HER3 with substantially the same orbetter affinity as the 2C2 antibody, comprising the 2C2 light chain (2C2VL (SEQ ID NO: 3) and the CL16 original heavy chain (SEQ ID NO: 2).

In one aspect of the present invention, an anti-HER3 antibody orantigen-binding fragment thereof specifically binds HER3 and antigenicfragments thereof with a dissociation constant of k_(d) (k_(off)/k_(on))of less than 10⁻⁶M, or of less than 10⁻⁷M, or of less than 10⁻⁸M, or ofless than 10⁻⁹M, or of less than 10⁻¹⁰ M, or of less than 10⁻¹¹M, or ofless than 10⁻¹² or of less than 10⁻¹³ M. In a particular aspect of thepresent invention, an anti-HER3 antibody or antigen-binding fragmentthereof specifically binds HER3 and antigenic fragments thereof with adissociation constant between 2×10⁻¹⁰ M and 6×10⁻¹⁰M.

In another aspect, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention binds to HER3 and/or antigenic fragmentsthereof with a K_(off) of less than 1×10⁻³ s ¹, or less than 2×10⁻³ s⁻¹.In other aspects, an anti-HER3 antibody or antigen-binding fragmentthereof binds to HER3 and antigenic fragments thereof with a K_(off) ofless than 10⁻³ s⁻¹, less than 5×10⁻³ s⁻¹, less than 10⁻⁴ s⁻¹, less than5×10⁻⁴ s⁻¹, less than 10⁻⁵ s⁻¹, less than 5×10⁻⁵ s⁻¹, less than 10⁻⁶s⁻¹, less than 5×10⁻⁶ s⁻¹, less than less than 5×10⁻⁷ s⁻¹, less than10⁻⁸ s⁻¹, less than 5×10⁻⁸ s⁻¹, less than 10⁻⁹ s⁻¹, less than 5×10⁻⁹s⁻¹, or less than 10⁻¹⁰ s⁻¹. In a particular aspect, an anti-HER3antibody or antigen-binding fragment thereof of the invention binds toHER3 and/or antigenic fragments thereof with a K_(off) of between0.5×10⁻⁴ s⁻¹ and 2.0×10⁻⁴ s⁻¹.

In another aspect, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention binds to HER3 and/or antigenic fragmentsthereof with an association rate constant or k_(on) rate of at least 10⁵M⁻¹ s⁻¹, at least 5×10⁵ M⁻¹ s⁻¹, at least 10⁶ M⁻¹ s⁻¹, at least 5×10⁶ M¹s¹, at least 10⁷ M¹ s¹, at least 5×10⁷ M¹ s¹, or at least 10⁸ M¹ s¹, orat least 10⁹M⁻¹ s⁻¹. In another aspect, an anti-HER3 antibody orantigen-binding fragment thereof of the invention binds to HER3 and/orantigenic fragments thereof with an association rate constant or k_(on)rate of between 1×10⁵ M⁻¹ s⁻¹ and 6×10⁵ M⁻¹ s⁻¹.

The VH and VL sequences disclosed in TABLE 1 can be “mixed and matched”to create other anti-HER3 binding molecules of the invention. In certainaspects, the VH sequences of 15D12.I and 15D12.V are mixed and matched.Additionally or alternatively, the VL sequences of 5H6, 8A3, 4H6, 6E.3,2B11, 2D1, 3A6, 4C4, 1A4, 2C2, 3E.1 can be mixed and matched.

In certain aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprises mutations that improve the binding tohuman FcRn and improve the half-life of the anti-HER3 antibody orantigen-binding fragment thereof. In some aspects, such mutations are amethionine (M) to tyrosine (Y) mutation in position 252, a serine (S) tothreonine (T) mutation in position 254, and a threonine (T) to glutamicacid (E) mutation in position 256, numbered according to the EU index asin Kabat (Kabat, et al. (1991) Sequences of Proteins of ImmunologicalInterest, U.S. Public Health Service, National Institutes of Health,Washington, D.C.), introduced into the constant domain of an IgG1. SeeU.S. Pat. No. 7,658,921, which is incorporated by reference herein. Thistype of mutant IgG, referred to as a “YTE mutant” has been shown displayapproximately four-times increased half-life as compared to wild-typeversions of the same antibody (Dall'Acqua et al., J. Biol. Chem.281:23514-24 (2006)). In some aspects, an anti-HER3 antibody orantigen-binding fragment thereof comprising an IgG constant domaincomprises one or more amino acid substitutions of amino acid residues atpositions 251-257, 285-290, 308-314, 385-389, and 428-436, numberedaccording to the EU index as in Kabat, wherein such mutations increasethe serum half-life of the anti-HER3 antibody or antigen-bindingfragment thereof.

In some aspects, a YTE mutant further comprises a substitution atposition 434 of the IgG constant domain, numbered according to the EUindex as in Kabat, with an amino acid selected from the group consistingof tryptophan (W), methionine (M), tyrosine (Y), and serine (S). Inother aspects, a YTE mutant further comprises a substitution at position434 of the IgG constant domain, numbered according to the EU index as inKabat, with an amino acid selected from the group consisting oftryptophan (W), methionine (M), tyrosine (Y), and serine (S), andsubstitution at position 428 of the IgG constant domain, numberedaccording to the EU index as in Kabat, with an amino acid selected fromthe group consisting of threonine (T), leucine (L), phenylalanine (F),and serine (S).

In yet other aspect, a YTE mutant further comprises a substitution atposition 434 of the IgG constant domain, numbered according to the EUindex as in Kabat, with tyrosine (Y), and a substitution at position 257of the IgG constant domain, numbered according to the EU index as inKabat, with leucine (L). In some aspects, a YTE mutant further comprisesa substitution at position 434 of the IgG constant domain, numberedaccording to the EU index as in Kabat, with serine (S), and asubstitution at position 428 of the IgG constant domain, numberedaccording to the EU index as in Kabat, with leucine (L).

In a specific aspect, an anti-HER3 antibody or antigen-binding fragmentthereof comprises a 2C2 light chain variable region (2C2 VL; SEQ ID NO:3), an original CL16 heavy chain variable region (SEQ ID NO: 2), and anIgG1 constant domain comprising a methionine (M) to tyrosine (Y)mutation in position 252, a serine (S) to threonine (T) mutation inposition 254, and a threonine (T) to glutamic acid (E) mutation inposition 256 of the IgG1 constant domain, numbered according to the EUindex as in Kabat.

In certain aspects, an anti-HER3 antibody or antigen-binding fragmentthereof of the invention comprise at least one IgG constant domain aminoacid substitution selected from the group consisting of:

-   -   (a) substitution of the amino acid at position 252 with tyrosine        (Y), phenylalanine (F), tryptophan (W), or threonine (T),    -   (b) substitution of the amino acid at position 254 with        threonine (T),    -   (c) substitution of the amino acid at position 256 with serine        (S), arginine (R), glutamine (Q), glutamic acid (E), aspartic        acid (D), or threonine (T),    -   (d) substitution of the amino acid at position 257 with leucine        (L),    -   (e) substitution of the amino acid at position 309 with proline        (P),    -   (f) substitution of the amino acid at position 311 with serine        (S),    -   (g) substitution of the amino acid at position 428 with        threonine (T), leucine (L), phenylalanine (F), or serine (S),    -   (h) substitution of the amino acid at position 433 with arginine        (R), serine (S), isoleucine (I), proline (P), or glutamine (Q),    -   (i) substitution of the amino acid at position 434 with        tryptophan (W), methionine (M), serine (S), histidine (H),        phenylalanine (F), or tyrosine, and    -   (j) a combination of two or more of said substitutions,

wherein the positions are numbered according to the EU index as inKabat, and

wherein the modified IgG has an increased serum half-life compared tothe serum half-life of an IgG having the wild-type IgG constant domain.

In other aspects, the VH and/or VL amino acid sequences can be 85%, 90%,95%, 96%, 97%, 98% or 99% similar to the sequences set forth above, andcomprise 1, 2, 3, 4, 5 or more conservative substitutions. A HER3antibody having VH and VL regions having high (i.e., 80% or greater)similarity to the VH regions of SEQ ID NOs: 2, 12 or 13 and/or VLregions of SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 16, or17, respectively, can be obtained by mutagenesis (e.g., site-directed orPCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ ID NOs:1-17, followed by testing of the encoded altered antibody for retainedfunction using the functional assays described herein.

The affinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method well known in the art, e.g.,flow cytometry, enzyme-linked immunosorbent assay (ELISA), orradioimmunoassay (RIA), or kinetics (e.g., BIACORE™ analysis). Directbinding assays as well as competitive binding assay formats can bereadily employed. (See, for example, Berzofsky et al., “Antibody-AntigenInteractions,” In Fundamental Immunology, Paul, W. E., Ed., Raven Press:New York, N.Y. (1984); Kuby, Immunology, W. H. Freeman and Company: NewYork, N.Y. (1992); and methods described herein. The measured affinityof a particular antibody-antigen interaction can vary if measured underdifferent conditions (e.g., salt concentration, pH, temperature). Thus,measurements of affinity and other antigen-binding parameters (e.g.,K_(D) or Kd, K_(on), K_(off)) are made with standardized solutions ofantibody and antigen, and a standardized buffer, as known in the art andsuch as the buffer described herein.

It also known in the art that affinities measured using BIACORE™analysis can vary depending on which one of the reactants is bound tothe chip. In this respect, affinity can be measured using a format inwhich the targeting antibody (e.g., the 2C2 monoclonal antibody) isimmobilized onto the chip (referred to as an “IgG down” format) or usinga format in which the target protein (e.g., HER3) is immobilized ontothe chip (referred to as, e.g., a “HER3 down” format).

III. Binding Molecules that Bind to the Same Epitope as anti-HER3Antibodies and Antigen-binding Fragments Thereof of the Invention

In another aspect, the invention comprises HER3-binding molecules thatbind to the same epitope as do the various anti-HER3 antibodiesdescribed herein. The term “epitope” as used herein refers to a proteindeterminant capable of binding to an antibody of the invention. Epitopesusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. Conformational and non-conformational epitopes aredistinguished in that the binding to the former but not the latter islost in the presence of denaturing solvents. Such antibodies can beidentified based on their ability to cross-compete (e.g., tocompetitively inhibit the binding of, in a statistically significantmanner) with antibodies such as the CL16 antibody, the 2C2 antibody, orthe 2C2-YTE mutant, in standard HER3 binding assays. Accordingly, in oneaspect, the invention provides anti-HER3 antibodies and antigen-bindingfragments thereof, e.g., human monoclonal antibodies, that compete forbinding to HER3 with another anti-HER3 antibody or antigen-bindingfragment thereof of the invention, such as the CL16 antibody or the 2C2antibody. The ability of a test antibody to inhibit the binding of,e.g., the CL16 antibody or the 2C2 antibody demonstrates that the testantibody can compete with that antibody for binding to HER3; such anantibody can, according to non-limiting theory, bind to the same or arelated (e.g., a structurally similar or spatially proximal) epitope onHER3 as the anti-HER3 antibody or antigen-binding fragment thereof withwhich it competes. In one aspect, the anti-HER3 antibody orantigen-binding fragment thereof that binds to the same epitope on HER3as, e.g., the CL16 antibody or the 2C2 antibody, is a human monoclonalantibody.

IV. Mechanism of Action

In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress HER3 phosphorylation. Inother aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress AKT phosphorylation. Instill other aspects, a HER3-binding molecule, e.g., an anti-HER3antibody or antigen-binding fragment thereof can suppress HER2-HER3dimer formation. In some aspects, a HER3-binding molecule, e.g., ananti-HER3 antibody or antigen-binding fragment thereof can suppress cellgrowth. In some aspects, a HER3-binding molecule, e.g., an anti-HER3antibody or antigen-binding fragment thereof lacks ADCC effect. Inspecific aspects, a HER3-binding molecule, e.g., an anti-HER3 antibodyor antigen-binding fragment thereof can suppress HER3 phosphorylation,AKT phosphorylation, and/or tumor colony formation via aligand-independent mechanism of action.

In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress HER3 phosphorylation inHRG-driven breast cancer MCF-7 cells as measured by ELISA, with an IC₅₀lower than about 30 ng/mL, lower than about 25 ng/mL, lower than about20 ng/mL, lower than about 15 ng/mL, or lower than about 10 ng/mL. In aspecific aspect, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress HER3 phosphorylation inHRG-driven breast cancer MCF-7 cells as measured by ELISA, with an IC₅₀lower than about 20 ng/mL. In a specific aspect, a HER3-bindingmolecule, e.g., an anti-HER3 antibody or antigen-binding fragmentthereof can suppress HER3 phosphorylation in HRG-driven breast cancerMCF-7 cells as measured by ELISA, with an IC₅₀ lower than about 15ng/mL. In another specific aspect, a HER3-binding molecule, e.g., ananti-HER3 antibody or antigen-binding fragment thereof can suppress HER3phosphorylation in HRG-driven breast cancer MCF-7 cells as measured byELISA, with an IC₅₀ lower than about 10 ng/mL.

In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress cell growth in MDA-MB-175breast cancer cells with an IC₅₀ lower than about 0.90 μg/mL, lower thanabout 0.80 μg/mL, lower than about 0.70 μg/mL, lower than about 0.60μg/mL, lower than about 0.50 μg/mL, lower than about 0.40 μg/mL, lowerthan about 0.30 μg/mL, or lower than about 0.20 μg/mL. In a specificaspect, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress cell growth in MDA-MB-175breast cancer cells, with an IC₅₀ lower than about 0.50 μg/mL. In aspecific aspect, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress cell growth in MDA-MB-175breast cancer cells, with an IC₅₀ lower than about 0.40 μg/mL. Inanother specific aspect, a HER3-binding molecule, e.g., an anti-HER3antibody or antigen-binding fragment thereof can suppress cell growth inMDA-MB-175 breast cancer cells, with an IC₅₀ lower than about 0.30μg/mL. In another specific aspect, a HER3-binding molecule, e.g., ananti-HER3 antibody or antigen-binding fragment thereof can suppress cellgrowth in MDA-MB-175 breast cancer cells, with an IC₅₀ lower than about0.20 μg/mL.

In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress cell growth in HMCBmelanoma cells with an IC₅₀ lower than about 0.20 μg/mL, lower thanabout 0.15 μg/mL, lower than about 0.10 μg/mL, lower than about 0.05μg/mL, lower than about 0.04 μg/mL, or lower than about 0.03 μg/mL. In aspecific aspect, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress cell growth in HMCBmelanoma cells, with an IC₅₀ lower than about 0.10 μg/mL. In a specificaspect, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress cell growth in HMCBmelanoma cells, with an IC₅₀ lower than about 0.05 μg/mL. In a specificaspect, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress cell growth in HMCBmelanoma cells, with an IC₅₀ lower than about 0.04 μg/mL. In a specificaspect, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress cell growth in HMCBmelanoma cells, with an IC₅₀ lower than about 0.03 μg/mL.

In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress HER3 phosphorylation inEGFR-driven HCC827 lung cancer cells with an IC₅₀ lower than about 20ng/mL, lower than about 15 ng/mL, lower than about 10 ng/mL, lower thanabout 8 ng/mL, lower than about 6 ng/mL, lower than about 4 ng/mL, orlower than about 2 ng/mL. In a specific aspect, a HER3-binding molecule,e.g., an anti-HER3 antibody or antigen-binding fragment thereof cansuppress HER3 phosphorylation in EGFR-driven HCC827 lung cancer cells,with an IC₅₀ lower than about 10 ng/mL. In a specific aspect, aHER3-binding molecule, e.g., an anti-HER3 antibody or antigen-bindingfragment thereof can suppress HER3 phosphorylation in EGFR-driven HCC827lung cancer cells, with an IC₅₀ lower than about 8 ng/mL. In a specificaspect, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress HER3 phosphorylation inEGFR-driven HCC827 lung cancer cells, with an IC₅₀ lower than about 6ng/mL. In a specific aspect, a HER3-binding molecule, e.g., an anti-HER3antibody or antigen-binding fragment thereof can suppress HER3phosphorylation in EGFR-driven HCC827 lung cancer cells, with an IC₅₀lower than about 4 ng/mL. In a specific aspect, a HER3-binding molecule,e.g., an anti-HER3 antibody or antigen-binding fragment thereof cansuppress HER3 phosphorylation in EGFR-driven HCC827 lung cancer cells,with an IC₅₀ lower than about 2 ng/mL.

In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress HER3 phosphorylation inEGFR-driven HCC827 lung cancer cells resistant to TKI with an IC₅₀ lowerthan about 30 ng/mL, lower than about 25 ng/mL, lower than about 20ng/mL, lower than about 15 ng/mL, lower than about 10 ng/mL, or lowerthan about 5 ng/mL. In a specific aspect, a HER3-binding molecule, e.g.,an anti-HER3 antibody or antigen-binding fragment thereof can suppressHER3 phosphorylation in EGFR-driven HCC827 lung cancer cells resistantto TKI, with an IC₅₀ lower than about 20 ng/mL. In a specific aspect, aHER3-binding molecule, e.g., an anti-HER3 antibody or antigen-bindingfragment thereof can suppress HER3 phosphorylation in EGFR-driven HCC827lung cancer cells resistant to TKI, with an IC₅₀ lower than about 15ng/mL. In a specific aspect, a HER3-binding molecule, e.g., an anti-HER3antibody or antigen-binding fragment thereof can suppress HER3phosphorylation in EGFR-driven HCC827 lung cancer cells resistant toTKI, with an IC₅₀ lower than about 10 ng/mL. In a specific aspect, aHER3-binding molecule, e.g., an anti-HER3 antibody or antigen-bindingfragment thereof can suppress HER3 phosphorylation in EGFR-driven HCC827lung cancer cells resistant to TKI, with an IC₅₀ lower than about 5ng/mL.

In some specific aspects, a HER3-binding molecule, e.g., an anti-HER3antibody or antigen-binding fragment thereof can be used to treat TKIresistant cancers.

In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress HER3 phosphorylation incMET-driven MKN45 human gastric adenocarcinoma cells with an IC₅₀ islower than about 15 ng/mL, lower than about 10 ng/mL, lower than about 9ng/mL, lower than about 8 ng/mL, lower than about 7 ng/mL, lower thanabout 6 ng/mL, lower than about 5 ng/mL, or lower than about 4 ng/mL. Ina specific aspect, a HER3-binding molecule, e.g., an anti-HER3 antibodyor antigen-binding fragment thereof can suppress HER3 phosphorylation incMET-driven MKN45 human gastric adenocarcinoma cells with an IC₅₀ lowerthan about 10 ng/mL. In a specific aspect, a HER3-binding molecule,e.g., an anti-HER3 antibody or antigen-binding fragment thereof cansuppress HER3 phosphorylation in cMET-driven MKN45 human gastricadenocarcinoma cells with an IC₅₀ lower than about 8 ng/mL. In aspecific aspect, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress HER3 phosphorylation incMET-driven MKN45 human gastric adenocarcinoma cells with an IC₅₀ lowerthan about 6 ng/mL. In a specific aspect, a HER3-binding molecule, e.g.,an anti-HER3 antibody or antigen-binding fragment thereof can suppressHER3 phosphorylation in cMET-driven MKN45 human gastric adenocarcinomacells with an IC₅₀ lower than about 4 ng/mL.

In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof of the invention can suppress pAKT incMET-driven MKN45 cells with an IC₅₀ lower than about 15 ng/mL, lowerthan about 10 ng/mL, lower than about 9 ng/mL, lower than about 8 ng/mL,lower than about 7 ng/mL, lower than about 6 ng/mL, lower than about 5ng/mL, lower than about 4 ng/mL, or lower than about 3 ng/mL. In aspecific aspect, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress pAKT in cMET-driven MKN45cells with an IC₅₀ lower than about 8 ng/mL. In a specific aspect, aHER3-binding molecule, e.g., an anti-HER3 antibody or antigen-bindingfragment thereof can suppress pAKT in cMET-driven MKN45 cells with anIC₅₀ lower than about 6 ng/mL. In a specific aspect, a HER3-bindingmolecule, e.g., an anti-HER3 antibody or antigen-binding fragmentthereof can suppress pAKT in cMET-driven MKN45 cells with an IC₅₀ lowerthan about 4 ng/mL. In a specific aspect, a HER3-binding molecule, e.g.,an anti-HER3 antibody or antigen-binding fragment thereof can suppresspAKT in cMET-driven MKN45 cells with an IC₅₀ lower than about 3 ng/mL.

In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof of the invention can suppress pHER inFGFR2-driven Kato III human gastric signet ring carcinoma cells with anIC₅₀ lower than about 9 ng/mL, lower than about 8 ng/mL, lower thanabout 7 ng/mL, lower than about 6 ng/mL, lower than about 5 ng/mL, lowerthan about 4 ng/mL, lower than about 3 ng/mL, lower than about 2 ng/mL,or lower than about 1 ng/mL. In a specific aspect, a HER3-bindingmolecule, e.g., an anti-HER3 antibody or antigen-binding fragmentthereof can suppress pHER in FGFR2-driven Kato III human gastric signetring carcinoma cells with an IC₅₀ lower than about 5 ng/mL. In aspecific aspect, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress pHER in FGFR2-driven KatoIII human gastric signet ring carcinoma cells with an IC₅₀ lower thanabout 4 ng/mL. In a specific aspect, a HER3-binding molecule, e.g., ananti-HER3 antibody or antigen-binding fragment thereof can suppress pHERin FGFR2-driven Kato III human gastric signet ring carcinoma cells withan IC₅₀ lower than about 3 ng/mL. In a specific aspect, a HER3-bindingmolecule, e.g., an anti-HER3 antibody or antigen-binding fragmentthereof can suppress pHER in FGFR2-driven Kato III human gastric signetring carcinoma cells with an IC₅₀ lower than about 2 ng/mL. In aspecific aspect, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress pHER in FGFR2-driven KatoIII human gastric signet ring carcinoma cells with an IC₅₀ lower thanabout 1 ng/mL.

In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress pAKT in FGFR-2 driven KatoIII cells with an IC₅₀ lower than about 6 ng/mL, lower than about 5ng/mL, lower than about 4 ng/mL, lower than about 3 ng/mL, lower thanabout 2 ng/mL, or lower than about 1 ng/mL. In a specific aspect, aHER3-binding molecule, e.g., an anti-HER3 antibody or antigen-bindingfragment thereof can suppress pAKT in FGFR-2 driven Kato III cells withan IC₅₀ lower than about 4 ng/mL. In a specific aspect, a HER3-bindingmolecule, e.g., an anti-HER3 antibody or antigen-binding fragmentthereof can suppress pAKT in FGFR-2 driven Kato III cells with an IC₅₀lower than about 3 ng/mL. In a specific aspect, a HER3-binding molecule,e.g., an anti-HER3 antibody or antigen-binding fragment thereof cansuppress pAKT in FGFR-2 driven Kato III cells with an IC₅₀ lower thanabout 2 ng/mL. In a specific aspect, a HER3-binding molecule, e.g., ananti-HER3 antibody or antigen-binding fragment thereof can suppress pAKTin FGFR-2 driven Kato III cells with an IC₅₀ lower than about 1 ng/mL.

In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof of the invention can suppress pHER inligand independent BT-474 breast cancer cells with an IC₅₀ lower thanabout 10 ng/mL, lower than about 9 ng/mL, lower than about 8 ng/mL,lower than about 7 ng/mL, lower than about 6 ng/mL, lower than about 5ng/mL, lower than about 4 ng/mL. In a specific aspect, a HER3-bindingmolecule, e.g., an anti-HER3 antibody or antigen-binding fragmentthereof can suppress pHER in ligand independent BT-474 breast cancercells with an IC₅₀ lower than about 8 ng/mL. In a specific aspect, aHER3-binding molecule, e.g., an anti-HER3 antibody or antigen-bindingfragment thereof can suppress pHER in ligand independent BT-474 breastcancer cells with an IC₅₀ lower than about 6 ng/mL. In a specificaspect, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress pHER in ligand independentBT-474 breast cancer cells with an IC₅₀ lower than about 4 ng/mL.

In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof of the invention can suppress pAKT inligand independent BT-474 breast cancer cells with an IC₅₀ lower thanabout 10 ng/mL, lower than about 9 ng/mL, lower than about 8 ng/mL,lower than about 7 ng/mL, lower than about 6 ng/mL, lower than about 5ng/mL, lower than about 4 ng/mL. In a specific aspect, a HER3-bindingmolecule, e.g., an anti-HER3 antibody or antigen-binding fragmentthereof can suppress pAKT in ligand independent BT-474 breast cancercells with an IC₅₀ lower than about 8 ng/mL. In a specific aspect, aHER3-binding molecule, e.g., an anti-HER3 antibody or antigen-bindingfragment thereof can suppress pAKT in ligand independent BT-474 breastcancer cells with an IC₅₀ lower than about 6 ng/mL. In a specificaspect, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress pAKT in ligand independentBT-474 breast cancer cells with an IC₅₀ lower than about 4 ng/mL. Insome aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof can suppress pHER3, pAKT, and tumorcolony formation in BT-474 cells, a ligand independent breast cancermodel.

In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof of the invention can suppress HRGinduced VEGF secretion. In a specific aspect, a HER3-binding molecule,e.g., an anti-HER3 antibody or antigen-binding fragment thereof of theinvention can suppress HRG induced VEGF secretion in ligand independentBT-474 breast cancer cells and/or HRG-driven breast cancer MCF-7 cells.

In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody orantigen-binding fragment thereof of the invention can cause cell cyclearrest. In a specific aspect, a HER3-binding molecule, e.g., ananti-HER3 antibody or antigen-binding fragment thereof of the inventioncan cause cell cycle arrest in breast cancer cells, including but notlimited to SKBR3 or BT474 cells.

V. Preparation of Anti-HER3 Antibodies and Antigen-Binding Fragments

Monoclonal anti-HER3 antibodies can be prepared using hybridoma methods,such as those described by Kohler and Milstein (1975) Nature 256:495.Using the hybridoma method, a mouse, hamster, or other appropriate hostanimal, is immunized as described above to elicit the production bylymphocytes of antibodies that will specifically bind to an immunizingantigen. Lymphocytes can also be immunized in vitro. Followingimmunization, the lymphocytes are isolated and fused with a suitablemyeloma cell line using, for example, polyethylene glycol, to formhybridoma cells that can then be selected away from unfused lymphocytesand myeloma cells. Hybridomas that produce monoclonal antibodiesdirected specifically against a chosen antigen as determined byimmunoprecipitation, immunoblotting, or by an in vitro binding assay(e.g. radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA))can then be propagated either in in vitro culture using standard methods(Goding, Monoclonal Antibodies: Principles and Practice, Academic Press,1986) or in vivo as ascites tumors in an animal. The monoclonalantibodies can then be purified from the culture medium or ascites fluidas described for polyclonal antibodies above.

Alternatively anti-HER3 monoclonal antibodies can also be made usingrecombinant DNA methods as described in U.S. Pat. No. 4,816,567. Thepolynucleotides encoding a monoclonal antibody are isolated from matureB-cells or hybridoma cell, such as by RT-PCR using oligonucleotideprimers that specifically amplify the genes encoding the heavy and lightchains of the antibody, and their sequence is determined usingconventional procedures. The isolated polynucleotides encoding the heavyand light chains are then cloned into suitable expression vectors, whichwhen transfected into host cells such as E. coli cells, simian COScells, Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant anti-HER3 monoclonalantibodies or antigen-binding fragments thereof of the desired speciescan be isolated from phage display libraries expressing CDRs of thedesired species as described (McCafferty et al., 1990, Nature,348:552-554; Clarkson et al., 1991, Nature, 352:624-628; and Marks etal., 1991, J. Mol. Biol., 222:581-597).

The polynucleotide(s) encoding a anti-HER3 antibody or antigen-bindingfragments thereof can further be modified in a number of differentmanners using recombinant DNA technology to generate alternativeantibodies. In some aspects, the constant domains of the light and heavychains of, for example, a mouse monoclonal antibody can be substituted(1) for those regions of, for example, a human antibody to generate achimeric antibody or (2) for a non-immunoglobulin polypeptide togenerate a fusion antibody. In some aspects, the constant regions aretruncated or removed to generate the desired antibody fragment of amonoclonal antibody. Site-directed or high-density mutagenesis of thevariable region can be used to optimize specificity, affinity, etc. of amonoclonal antibody.

In certain aspects, the anti-HER3 antibody or antigen-binding fragmentthereof is a human antibody or antigen-binding fragment thereof. Humanantibodies can be directly prepared using various techniques known inthe art. Immortalized human B lymphocytes immunized in vitro or isolatedfrom an immunized individual that produce an antibody directed against atarget antigen can be generated (See, e.g., Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer etal., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat. No. 5,750,373).

Also, the anti-HER3 human antibody or antigen-binding fragment thereofcan be selected from a phage library, where that phage library expresseshuman antibodies, as described, for example, in Vaughan et al., 1996,Nat. Biotech., 14:309-314, Sheets et al., 1998, Proc. Nat'l. Acad. Sci.,95:6157-6162, Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381, andMarks et al., 1991, J. Mol. Biol., 222:581). Techniques for thegeneration and use of antibody phage libraries are also described inU.S. Pat. Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404; 6,544,731;6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484; and7,264,963; and Rothe et al., 2007, J. Mol. Bio.,doi:10.1016/j.jmb.2007.12.018 (each of which is incorporated byreference in its entirety).

Affinity maturation strategies and chain shuffling strategies (Marks etal., 1992, Bio/Technology 10:779-783, incorporated by reference in itsentirety) are known in the art and can be employed to generate highaffinity human antibodies or antigen-binding fragments thereof.

In some aspects, an anti-HER3 monoclonal antibody can be a humanizedantibody. Methods for engineering, humanizing or resurfacing non-humanor human antibodies can also be used and are well known in the art. Ahumanized, resurfaced or similarly engineered antibody can have one ormore amino acid residues from a source that is non-human, e.g., but notlimited to, mouse, rat, rabbit, non-human primate or other mammal. Thesenon-human amino acid residues are replaced by residues that are oftenreferred to as “import” residues, which are typically taken from an“import” variable, constant or other domain of a known human sequence.Such imported sequences can be used to reduce immunogenicity or reduce,enhance or modify binding, affinity, on-rate, off-rate, avidity,specificity, half-life, or any other suitable characteristic, as knownin the art. In general, the CDR residues are directly and mostsubstantially involved in influencing HER3 binding. Accordingly, part orall of the non-human or human CDR sequences are maintained while thenon-human sequences of the variable and constant regions can be replacedwith human or other amino acids.

Antibodies can also optionally be humanized, resurfaced, engineered orhuman antibodies engineered with retention of high affinity for theantigen HER3 and other favorable biological properties. To achieve thisgoal, humanized (or human) or engineered anti-HER3 antibodies andresurfaced antibodies can be optionally prepared by a process ofanalysis of the parental sequences and various conceptual humanized andengineered products using three-dimensional models of the parental,engineered, and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen, such as HER3. In this way, framework (FW) residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved.

Humanization, resurfacing or engineering of anti-HER3 antibodies orantigen-binding fragments thereof of the present invention can beperformed using any known method, such as but not limited to thosedescribed in, Jones et al., Nature 321:522 (1986); Riechmann et al.,Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), Simset al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol.196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285(1992); Presta et al., J. Immunol. 151:2623 (1993), U.S. Pat. Nos.5,639,641, 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476;5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370;5,693,762; 5,530,101; 5,585,089; 5,225,539; 4,816,567, 7,557,189;7,538,195; and 7,342,110; International Application Nos. PCT/US98/16280;PCT/US96/18978; PCT/US91/09630; PCT/US91/05939; PCT/US94/01234;PCT/GB89/01334; PCT/GB91/01134; PCT/GB92/01755; International PatentApplication Publication Nos. WO90/14443; WO90/14424; WO90/14430; andEuropean Patent Publication No. EP 229246; each of which is entirelyincorporated herein by reference, including the references citedtherein.

Anti-HER3 humanized antibodies and antigen-binding fragments thereof canalso be made in transgenic mice containing human immunoglobulin locithat are capable upon immunization of producing the full repertoire ofhuman antibodies in the absence of endogenous immunoglobulin production.This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; and 5,661,016.

In certain aspects an anti-HER3 antibody fragment is provided. Varioustechniques are known for the production of antibody fragments.Traditionally, these fragments are derived via proteolytic digestion ofintact antibodies (for example Morimoto et al., 1993, Journal ofBiochemical and Biophysical Methods 24:107-117; Brennan et al., 1985,Science, 229:81). In certain aspects, anti-HER3 antibody fragments areproduced recombinantly. Fab, Fv, and scFv antibody fragments can all beexpressed in and secreted from E. coli or other host cells, thusallowing the production of large amounts of these fragments. Suchanti-HER3 antibody fragments can also be isolated from the antibodyphage libraries discussed above. The anti-HER3 antibody fragments canalso be linear antibodies as described in U.S. Pat. No. 5,641,870. Othertechniques for the production of antibody fragments will be apparent tothe skilled practitioner.

According to the present invention, techniques can be adapted for theproduction of single-chain antibodies specific to HER3 (see, e.g., U.S.Pat. No. 4,946,778). In addition, methods can be adapted for theconstruction of Fab expression libraries (see, e.g., Huse et al.,Science 246:1275-1281 (1989)) to allow rapid and effectiveidentification of monoclonal Fab fragments with the desired specificityfor HER3, or derivatives, fragments, analogs or homologs thereofAntibody fragments can be produced by techniques in the art including,but not limited to: (a) a F(ab′)2 fragment produced by pepsin digestionof an antibody molecule; (b) a Fab fragment generated by reducing thedisulfide bridges of an F(ab′)2 fragment, (c) a Fab fragment generatedby the treatment of the antibody molecule with papain and a reducingagent, and (d) Fv fragments.

It can further be desirable, especially in the case of antibodyfragments, to modify an anti-HER3 antibody or antigen-binding fragmentthereof in order to increase its serum half-life. This can be achieved,for example, by incorporation of a salvage receptor binding epitope intothe antibody or antibody fragment by mutation of the appropriate regionin the antibody or antibody fragment or by incorporating the epitopeinto a peptide tag that is then fused to the antibody or antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis), or by YTE mutation. Other methods to increase the serumhalf-life of an antibody or antigen-binding fragment thereof, e.g.,conjugation to a heterologous molecule such as PEG are known in the art.

Heteroconjugate anti-HER3 antibodies and antigen-binding fragmentsthereof are also within the scope of the present invention.Heteroconjugate antibodies are composed of two covalently joinedantibodies. Such antibodies have, for example, been proposed to targetimmune cells to unwanted cells (see, e.g., U.S. Pat. No. 4,676,980). Itis contemplated that the heteroconjugate anti-HER3 antibodies andantigen-binding fragments thereof can be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins can be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

In certain aspects, the HER3-binding molecules of the invention, e.g.,antibodies or antigen-binding fragments thereof can be combined withother therapeutic agents or conjugated to other therapeutic agents ortoxins to form immunoconjugates and/or fusion proteins. Examples of suchtherapeutic agents and toxins include, but are not limited to cetuximab(Erbitux®), panitumumab (Vectibix®), lapatinib (Tykerb®/Tyverb®), andpaclitaxel (Taxol®, Abraxane®) and derivatives (e.g., docetaxel).

In some aspects the HER3-binding molecules of the invention, e.g.,antibodies or antigen-binding fragments thereof can be conjugated toantibodies or antibody fragments targeting epidermal growth factorreceptor (EGFR). In other aspects, the HER3-binding molecules of theinvention can be conjugated to tyrosine kinase inhibitors. In somespecific aspects, the HER3-binding molecules of the invention can beconjugated to inhibitors of the tyrosine kinase activity associated withEGFR and/or HER2/neu. In some aspects, the HER3-binding molecules of theinvention can be conjugated to antimitotic agents. In some specificaspects, the HER3-binding molecules of the invention can be conjugatedto agents that stabilize the mitotic spindle microtubule assembly.

For the purposes of the present invention, it should be appreciated thatmodified anti-HER3 antibodies or antigen-binding fragments thereof cancomprise any type of variable region that provides for the associationof the antibody or polypeptide with HER3. In this regard, the variableregion can comprise or be derived from any type of mammal that can beinduced to mount a humoral response and generate immunoglobulins againstthe desired tumor associated antigen. As such, the variable region ofthe modified anti-HER3 antibodies or antigen-binding fragments thereofcan be, for example, of human, murine, non-human primate (e.g.,cynomolgus monkeys, macaques, etc.) or lupine origin. In some aspectsboth the variable and constant regions of the modified anti-HER3antibodies or antigen-binding fragments thereof are human. In otheraspects the variable regions of compatible antibodies (usually derivedfrom a non-human source) can be engineered or specifically tailored toimprove the binding properties or reduce the immunogenicity of themolecule. In this respect, variable regions useful in the presentinvention can be humanized or otherwise altered through the inclusion ofimported amino acid sequences.

In certain aspects, the variable domains in both the heavy and lightchains of an anti-HER3 antibody or antigen-binding fragment thereof arealtered by at least partial replacement of one or more CDRs and, ifnecessary, by partial framework region replacement and sequencechanging. Although the CDRs can be derived from an antibody of the sameclass or even subclass as the antibody from which the framework regionsare derived, it is envisaged that the CDRs will be derived from anantibody of different class and in certain aspects from an antibody froma different species. It is not necessary to replace all of the CDRs withthe complete CDRs from the donor variable region to transfer the antigenbinding capacity of one variable domain to another. Rather, it is onlynecessary to transfer those residues that are necessary to maintain theactivity of the antigen binding site. Given the explanations set forthin U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, it will be wellwithin the competence of those skilled in the art, either by carryingout routine experimentation or by trial and error testing to obtain afunctional antibody with reduced immunogenicity.

Alterations to the variable region notwithstanding, those skilled in theart will appreciate that the modified anti-HER3 antibodies orantigen-binding fragments thereof of this invention will compriseantibodies (e.g., full-length antibodies or immunoreactive fragmentsthereof) in which at least a fraction of one or more of the constantregion domains has been deleted or otherwise altered so as to providedesired biochemical characteristics such as increased tumor localizationor reduced serum half-life when compared with an antibody ofapproximately the same immunogenicity comprising a native or unalteredconstant region. In some aspects, the constant region of the modifiedantibodies will comprise a human constant region. Modifications to theconstant region compatible with this invention comprise additions,deletions or substitutions of one or more amino acids in one or moredomains. That is, the modified antibodies disclosed herein can comprisealterations or modifications to one or more of the three heavy chainconstant domains (CH1, CH2 or CH3) and/or to the light chain constantdomain (CL). In some aspects, modified constant regions wherein one ormore domains are partially or entirely deleted are contemplated. In someaspects, the modified antibodies will comprise domain deleted constructsor variants wherein the entire CH2 domain has been removed (ACH2constructs). In some aspects, the omitted constant region domain will bereplaced by a short amino acid spacer (e.g., 10 residues) that providessome of the molecular flexibility typically imparted by the absentconstant region.

Besides their configuration, it is known in the art that the constantregion mediates several effector functions. For example, binding of theC1 component of complement to antibodies activates the complementsystem. Activation of complement is important in the opsonisation andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and can also be involved in autoimmunehypersensitivity. Further, antibodies bind to cells via the Fc region,with a Fc receptor site on the antibody Fc region binding to a Fcreceptor (FcR) on a cell. There are a number of Fc receptors which arespecific for different classes of antibody, including IgG (gammareceptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mureceptors). Binding of antibody to Fc receptors on cell surfacestriggers a number of important and diverse biological responsesincluding engulfment and destruction of antibody-coated particles,clearance of immune complexes, lysis of antibody-coated target cells bykiller cells (called antibody-dependent cell-mediated cytotoxicity, orADCC), release of inflammatory mediators, placental transfer and controlof immunoglobulin production.

In certain aspects, a anti-HER3 antibody or an antigen-binding fragmentthereof provides for altered effector functions that, in turn, affectthe biological profile of the administered antibody or antigen-bindingfragment thereof. For example, the deletion or inactivation (throughpoint mutations or other means) of a constant region domain can reduceFc receptor binding of the circulating modified antibody therebyincreasing tumor localization. In other cases it can be that constantregion modifications, consistent with this invention, moderatecomplement binding and thus reduce the serum half-life and nonspecificassociation of a conjugated cytotoxin. Yet other modifications of theconstant region can be used to eliminate disulfide linkages oroligosaccharide moieties that allow for enhanced localization due toincreased antigen specificity or antibody flexibility. Similarly,modifications to the constant region in accordance with this inventioncan easily be made using well known biochemical or molecular engineeringtechniques well within the purview of the skilled artisan.

In certain aspects, a HER3-binding molecule that is an antibody orantigen-binding fragment thereof does not have one or more effectorfunctions. For instance, in some aspects, the antibody orantigen-binding fragment thereof has no antibody-dependent cellularcytotoxicity (ADCC) activity and/or no complement-dependent cytotoxicity(CDC) activity. In certain aspects, the anti-HER3 antibody or antigenbinding fragment thereof does not bind to an Fc receptor and/orcomplement factors. In certain aspects, the antibody or antigen-bindingfragment thereof has no effector function.

It will be noted that in certain aspects, the anti-HER3 modifiedantibodies or antigen-binding fragments thereof can be engineered tofuse the CH3 domain directly to the hinge region of the respectivemodified antibodies or fragments thereof. In other constructs it can bedesirable to provide a peptide spacer between the hinge region and themodified CH2 and/or CH3 domains. For example, compatible constructscould be expressed wherein the CH2 domain has been deleted and theremaining CH3 domain (modified or unmodified) is joined to the hingeregion with a 5-20 amino acid spacer. Such a spacer can be added, forinstance, to ensure that the regulatory elements of the constant domainremain free and accessible or that the hinge region remains flexible.However, it should be noted that amino acid spacers can, in some cases,prove to be immunogenic and elicit an unwanted immune response againstthe construct. Accordingly, in certain aspects, any spacer added to theconstruct will be relatively non-immunogenic, or even omittedaltogether, so as to maintain the desired biochemical qualities of themodified antibodies.

Besides the deletion of whole constant region domains, it will beappreciated that the anti-HER3 antibodies and antigen-binding fragmentsthereof of the present invention can be provided by the partial deletionor substitution of a few or even a single amino acid. For example, themutation of a single amino acid in selected areas of the CH2 domain canbe enough to substantially reduce Fc binding and thereby increase tumorlocalization. Similarly, it can be desirable to simply delete that partof one or more constant region domains that control the effectorfunction (e.g., complement C1Q binding) to be modulated. Such partialdeletions of the constant regions can improve selected characteristicsof the antibody or antigen-binding fragment thereof (e.g., serumhalf-life) while leaving other desirable functions associated with thesubject constant region domain intact. Moreover, as alluded to above,the constant regions of the disclosed anti-HER3 antibodies andantigen-binding fragments thereof can be modified through the mutationor substitution of one or more amino acids that enhances the profile ofthe resulting construct. In this respect it is possible to disrupt theactivity provided by a conserved binding site (e.g., Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody or antigen-binding fragment thereof. Certainaspects can comprise the addition of one or more amino acids to theconstant region to enhance desirable characteristics such as decreasingor increasing effector function or provide for more cytotoxin orcarbohydrate attachment. In such aspects it can be desirable to insertor replicate specific sequences derived from selected constant regiondomains.

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized and humananti-HER3 antibodies, or antigen-binding fragments thereof, set forthherein. These can contain, for example, conservative substitutionmutations, i.e., the substitution of one or more amino acids by similaramino acids. For example, conservative substitution refers to thesubstitution of an amino acid with another within the same general classsuch as, for example, one acidic amino acid with another acidic aminoacid, one basic amino acid with another basic amino acid or one neutralamino acid by another neutral amino acid. What is intended by aconservative amino acid substitution is well known in the art.

An anti-HER3 antibody or antigen-binding fragment thereof can be furthermodified to contain additional chemical moieties not normally part ofthe protein. Those derivatized moieties can improve the solubility, thebiological half-life or absorption of the protein. The moieties can alsoreduce or eliminate any desirable side effects of the proteins and thelike. An overview for those moieties can be found in Remington'sPharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, Pa.(2000).

VI. Polynucleotides Encoding HER3-Binding Molecules

In certain aspects, the invention encompasses polynucleotides comprisingnucleic acid sequences that encode a polypeptide that specifically bindsHER3 or an antigen-binding fragment thereof. For example, the inventionprovides a polynucleotide comprising a nucleic acid sequence thatencodes an anti-HER3 antibody or encodes an antigen-binding fragment ofsuch an antibody. The polynucleotides of the invention can be in theform of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, andsynthetic DNA; and can be double-stranded or single-stranded, and ifsingle stranded can be the coding strand or non-coding (anti-sense)strand.

In certain aspects, the polynucleotides are isolated. In certainaspects, the polynucleotides are substantially pure. In certain aspectsthe polynucleotides comprise the coding sequence for the maturepolypeptide fused in the same reading frame to a polynucleotide whichaids, for example, in expression and secretion of a polypeptide from ahost cell (e.g., a leader sequence which functions as a secretorysequence for controlling transport of a polypeptide from the cell). Thepolypeptide having a leader sequence is a preprotein and can have theleader sequence cleaved by the host cell to form the mature form of thepolypeptide. The polynucleotides can also encode for an HER3-bindingproprotein which is the mature protein plus additional 5′ amino acidresidues.

In certain aspects the polynucleotides comprise the coding sequence forthe mature HER3-binding polypeptide, e.g., an anti-HER3 antibody or anantigen-binding fragment thereof fused in the same reading frame to amarker sequence that allows, for example, for purification of theencoded polypeptide. For example, the marker sequence can be ahexa-histidine tag supplied by a pQE-9 vector to provide forpurification of the mature polypeptide fused to the marker in the caseof a bacterial host, or the marker sequence can be a hemagglutinin (HA)tag derived from the influenza hemagglutinin protein when a mammalianhost (e.g., COS-7 cells) is used.

The present invention further relates to variants of the describedpolynucleotides encoding, for example, HER3-binding fragments, analogs,and derivatives of the HER3-binding molecules of the invention.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some aspects the polynucleotidevariants contain alterations which produce silent substitutions,additions, or deletions, but do not alter the properties or activitiesof the encoded polypeptide. In some aspects, nucleotide variants areproduced by silent substitutions due to the degeneracy of the geneticcode. Polynucleotide variants can be produced for a variety of reasons,e.g., to optimize codon expression for a particular host (change codonsin the human mRNA to those preferred by a bacterial host such as E.coli). Vectors and cells comprising the polynucleotides described hereinare also provided.

In some aspects a DNA sequence encoding a HER3-binding molecule, e.g.,an anti-HER3 antibody or an antigen-binding fragment thereof can beconstructed by chemical synthesis using an oligonucleotide synthesizer.Such oligonucleotides can be designed based on the amino acid sequenceof the desired polypeptide and selecting those codons that are favoredin the host cell in which the recombinant polypeptide of interest willbe produced. Standard methods can be applied to synthesize an isolatedpolynucleotide sequence encoding an isolated polypeptide of interest.For example, a complete amino acid sequence can be used to construct aback-translated gene. Further, a DNA oligomer containing a nucleotidesequence coding for the particular isolated polypeptide can besynthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the polynucleotide sequences encoding a particular isolatedpolypeptide of interest will be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the protein in a desired host. Proper assembly can beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene must be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

In certain aspects, recombinant expression vectors are used to amplifyand express DNA encoding anti-HER3 antibodies or antigen-bindingfragments thereof. Recombinant expression vectors are replicable DNAconstructs which have synthetic or cDNA-derived DNA fragments encoding apolypeptide chain of an anti-HER3 antibody or and antigen-bindingfragment thereof, operatively linked to suitable transcriptional ortranslational regulatory elements derived from mammalian, microbial,viral or insect genes. A transcriptional unit generally comprises anassembly of (1) a genetic element or elements having a regulatory rolein gene expression, for example, transcriptional promoters or enhancers,(2) a structural or coding sequence which is transcribed into mRNA andtranslated into protein, and (3) appropriate transcription andtranslation initiation and termination sequences, as described in detailbelow. Such regulatory elements can include an operator sequence tocontrol transcription. The ability to replicate in a host, usuallyconferred by an origin of replication, and a selection gene tofacilitate recognition of transformants can additionally beincorporated. DNA regions are operatively linked when they arefunctionally related to each other. For example, DNA for a signalpeptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. Structural elements intendedfor use in yeast expression systems include a leader sequence enablingextracellular secretion of translated protein by a host cell.Alternatively, where recombinant protein is expressed without a leaderor transport sequence, it can include an N-terminal methionine residue.This residue can optionally be subsequently cleaved from the expressedrecombinant protein to provide a final product.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations can be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from E. coli, including pCR1, pBR322, pMB9 and their derivatives, wider host range plasmids, suchas M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of an HER3-binding molecule, e.g., ananti-HER3 antibody or antigen-binding fragment thereof includeprokaryotes, yeast, insect or higher eukaryotic cells under the controlof appropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Higher eukaryoticcells include established cell lines of mammalian origin as describedbelow. Cell-free translation systems could also be employed. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described by Pouwels et al. (CloningVectors: A Laboratory Manual, Elsevier, N.Y., 1985), the relevantdisclosure of which is hereby incorporated by reference. Additionalinformation regarding methods of protein production, including antibodyproduction, can be found, e.g., in U.S. Patent Publication No.2008/0187954, U.S. Pat. Nos. 6,413,746 and 6,660,501, and InternationalPatent Publication No. WO 04009823, each of which is hereby incorporatedby reference herein in its entirety.

Various mammalian or insect cell culture systems can also beadvantageously employed to express recombinant HER3-binding molecules,e.g., anti-HER3 antibodies or antigen-binding fragments thereof.Expression of recombinant proteins in mammalian cells can be performedbecause such proteins are generally correctly folded, appropriatelymodified and completely functional. Examples of suitable mammalian hostcell lines include HEK-293 and HEK-293T, the COS-7 lines of monkeykidney cells, described by Gluzman (Cell 23:175, 1981), and other celllines including, for example, L cells, C127, 3T3, Chinese hamster ovary(CHO), NSO, HeLa and BHK cell lines. Mammalian expression vectors cancomprise nontranscribed elements such as an origin of replication, asuitable promoter and enhancer linked to the gene to be expressed, andother 5′ or 3′ flanking nontranscribed sequences, and 5′ or 3′nontranslated sequences, such as necessary ribosome binding sites, apolyadenylation site, splice donor and acceptor sites, andtranscriptional termination sequences. Baculovirus systems forproduction of heterologous proteins in insect cells are reviewed byLuckow and Summers, BioTechnology 6:47 (1988).

HER3-binding molecules, e.g., anti-HER3 antibodies or antigen-bindingfragments thereof produced by a transformed host can be purifiedaccording to any suitable method. Such standard methods includechromatography (e.g., ion exchange, affinity and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for protein purification. Affinity tags such ashexahistidine, maltose binding domain, influenza coat sequence andglutathione-S-transferase can be attached to the protein to allow easypurification by passage over an appropriate affinity column. Isolatedproteins can also be physically characterized using such techniques asproteolysis, nuclear magnetic resonance and x-ray crystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify an HER3-binding molecule. Some or all of theforegoing purification steps, in various combinations, can also beemployed to provide a homogeneous recombinant protein.

A recombinant HER3-binding protein, e.g., an anti-HER3 antibody orantigen-binding fragment thereof produced in bacterial culture can beisolated, for example, by initial extraction from cell pellets, followedby one or more concentration, salting-out, aqueous ion exchange or sizeexclusion chromatography steps. High performance liquid chromatography(HPLC) can be employed for final purification steps. Microbial cellsemployed in expression of a recombinant protein can be disrupted by anyconvenient method, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Methods known in the art for purifying antibodies and other proteinsalso include, for example, those described in U.S. Patent PublicationNos. 2008/0312425, 2008/0177048, and 2009/0187005, each of which ishereby incorporated by reference herein in its entirety.

In certain aspects, the HER3-binding molecule is a polypeptide that isnot an antibody. A variety of methods for identifying and producingnon-antibody polypeptides that bind with high affinity to a proteintarget are known in the art. See, e.g., Skerra, Curr. Opin. Biotechnol.,18:295-304 (2007), Hosse et al., Protein Science, 15:14-27 (2006), Gillet al., Curr. Opin. Biotechnol., 17:653-658 (2006), Nygren, FEBS J.,275:2668-76 (2008), and Skerra, FEBS J., 275:2677-83 (2008), each ofwhich is incorporated by reference herein in its entirety. In certainaspects, phage display technology can been used to identify/produce anHER3-binding polypeptide. In certain aspects, the polypeptide comprisesa protein scaffold of a type selected from the group consisting ofprotein A, a lipocalin, a fibronectin domain, an ankyrin consensusrepeat domain, and thioredoxin.

VI. Treatment Methods Using Therapeutic Anti-HER3 Antibodies

Methods of the invention are directed to the use of anti-HER3 bindingmolecules, e.g., antibodies, including antigen-binding fragments,variants, and derivatives thereof, to treat patients having a diseaseassociated with HER3 expression or HER3-expressing cells. By“HER3-expressing cell” is meant a cell expressing HER3. Methods fordetecting HER3 expression in cells are well known in the art andinclude, but are not limited to, PCR techniques, immunohistochemistry,flow cytometry, Western blot, ELISA, and the like.

Though the following discussion refers to diagnostic methods andtreatment of various diseases and disorders with an HER3-bindingmolecule of the invention, the methods described herein are alsoapplicable to anti-HER3 antibodies, and the antigen-binding fragments,variants, and derivatives of these anti-HER3 antibodies that retain thedesired properties of the anti-HER3 antibodies of the invention, e.g.,capable of specifically binding HER3 and neutralizing HER3 activity. Insome aspects, HER3-binding molecules are human or humanized antibodiesthat do not mediate human ADCC, or are selected from known anti-HER3antibodies that do not mediate ADCC, or are anti-HER3 antibodies thatare engineered such that they do not mediate ADCC. In some aspects, theHER3-binding molecule is a clone 16 monoclonal antibody. In otheraspects, the HER3-binding molecule is a clone 16 YTE mutant antibody. Insome aspects the HER3-binding molecule is a P2B11 monoclonal antibody.In some aspects the HER3-binding molecule is a 1A4 monoclonal antibody.In some aspects the HER3-binding molecule is a 2C2 monoclonal antibody.In some aspects the HER3-binding molecule is a 2F10 monoclonal antibody.In some aspects the HER3-binding molecule is a 3E1 monoclonal antibody.In some aspects the HER3-binding molecule is a P2B 11 monoclonalantibody engineered to extend serum half-life. In some aspects theHER3-binding molecule is a 1A4 monoclonal antibody engineered to extendserum half-life. In some aspects the HER3-binding molecule is a 2C2monoclonal antibody engineered to extend serum half-life. In someaspects the HER3-binding molecule is a 2F10 monoclonal antibodyengineered to extend serum half-life. In some aspects the HER3-bindingmolecule is a 3E1 monoclonal antibody engineered to extend serumhalf-life. In other aspects the HER3-binding molecule is a P2B11 YTEmutant antibody. In other aspects the HER3-binding molecule is a 1A4 YTEmutant antibody. In other aspects the HER3-binding molecule is a 2C2-YTEmutant antibody. In other aspects the HER3-binding molecule is a 2F10YTE mutant antibody. In other aspects the HER3-binding molecule is a 3E1YTE mutant antibody.

In one aspect, treatment includes the application or administration ofan anti-HER3 binding molecule, e.g., an antibody or antigen bindingfragment, variant, or derivative thereof of the current invention to asubject or patient, or application or administration of the anti-HER3binding molecule to an isolated tissue or cell line from a subject orpatient, where the subject or patient has a disease, a symptom of adisease, or a predisposition toward a disease. In another aspect,treatment is also intended to include the application or administrationof a pharmaceutical composition comprising the anti-HER3 bindingmolecule, e.g., an antibody or antigen binding fragment, variant, orderivative thereof of the current invention to a subject or patient, orapplication or administration of a pharmaceutical composition comprisingthe anti-HER3 binding molecule to an isolated tissue or cell line from asubject or patient, who has a disease, a symptom of a disease, or apredisposition toward a disease.

The anti-HER3 binding molecules, e.g., antibodies or antigen-bindingfragments, variants, or derivatives thereof of the present invention areuseful for the treatment of various cancers. In one aspect, theinvention relates to anti-HER binding molecules, e.g., antibodies orantigen-binding fragments, variants, or derivatives thereof for use as amedicament, in particular for use in the treatment or prophylaxis ofcancer. Examples of cancer include, but are not limited to colon cancer,lung cancer, gastric cancer, head and neck squamous cells cancer,melanoma, pancreatic cancer, prostate cancer, and breast cancer.

In accordance with the methods of the present invention, at least oneanti-HER3 binding molecule, e.g., an antibody or antigen bindingfragment, variant, or derivative thereof as defined elsewhere herein isused to promote a positive therapeutic response with respect to cancer.The term “positive therapeutic response” with respect to cancertreatment refers to an improvement in the disease in association withthe activity of these anti-HER3 binding molecules, e.g., antibodies orantigen-binding fragments, variants, or derivatives thereof, and/or animprovement in the symptoms associated with the disease. Thus, forexample, an improvement in the disease can be characterized as acomplete response. By “complete response” is intended an absence ofclinically detectable disease with normalization of any previously testresults. Alternatively, an improvement in the disease can be categorizedas being a partial response. A “positive therapeutic response”encompasses a reduction or inhibition of the progression and/or durationof cancer, the reduction or amelioration of the severity of cancer,and/or the amelioration of one or more symptoms thereof resulting fromthe administration of an anti-HER3 binding molecule of the invention. Inspecific aspects, such terms refer to one, two or three or more resultsfollowing the administration of anti-HER3 binding molecules of theinvention: (1) a stabilization, reduction or elimination of the cancercell population; (2) a stabilization or reduction in cancer growth; (3)an impairment in the formation of cancer; (4) eradication, removal, orcontrol of primary, regional and/or metastatic cancer; (5) a reductionin mortality; (6) an increase in disease-free, relapse-free,progression-free, and/or overall survival, duration, or rate; (7) anincrease in the response rate, the durability of response, or number ofpatients who respond or are in remission; (8) a decrease inhospitalization rate, (9) a decrease in hospitalization lengths, (10)the size of the cancer is maintained and does not increase or increasesby less than 10%, preferably less than 5%, preferably less than 4%,preferably less than 2%, and (12) an increase in the number of patientsin remission.

Clinical response can be assessed using screening techniques such asmagnetic resonance imaging (MRI) scan, x-radiographic imaging, computedtomographic (CT) scan, flow cytometry or fluorescence-activated cellsorter (FACS) analysis, histology, gross pathology, and blood chemistry,including but not limited to changes detectable by ELISA, RIA,chromatography, and the like. In addition to these positive therapeuticresponses, the subject undergoing therapy with the anti-HER3 bindingmolecule, e.g., an antibody or antigen-binding fragment, variant, orderivative thereof, can experience the beneficial effect of animprovement in the symptoms associated with the disease.

The anti-HER3 binding molecules, e.g., antibodies or antigen-bindingfragments, variants, or derivatives thereof of the invention can be usedin combination with any known therapies for cancer, including any agentor combination of agents that are known to be useful, or which have beenused or are currently in use, for treatment of cancer, e.g., coloncancer, lung cancer, gastric cancer, head and neck squamous cellscancer, and breast cancer. The second agent or combination of agents ofthe pharmaceutical combination formulation or dosing regimen preferablyhas complementary activities to the antibody or polypeptide of theinvention such that they do not adversely affect each other.

Anticancer agents include drugs used to treat malignancies, such ascancerous growths. Drug therapy can be used alone, or in combinationwith other treatments such as surgery or radiation therapy. Severalclasses of drugs can be used in cancer treatment, depending on thenature of the organ involved. For example, breast cancers are commonlystimulated by estrogens, and can be treated with drugs which inactivethe sex hormones. Similarly, prostate cancer can be treated with drugsthat inactivate androgens, the male sex hormone. Anti-cancer agents foruse in certain methods of the present invention include, among others,antibodies (e.g., antibodies which bind IGF-1R, antibodies which bindEGFR, antibodies which bind HER2, antibodies which bind HER3, orantibodies which bind cMET), small molecules targeting IGF1R, smallmolecules targeting EGFR, small molecules targeting HER2,antimetabolites, alkylating agents, topoisomerase inhibitors,microtubule targeting agents, kinase inhibitors, protein synthesisinhibitors, immunotherapeutic agents, hormonal therapies,glucocorticoids, aromatase inhibitors, mTOR inhibitors, chemotherapeuticagents, Protein Kinase B inhibitors, Phosphatidylinositol 3-Kinase(PI3K) inhibitors, Cyclin Dependent Kinase (CDK) inhibitors, RLr9,CD289, enzyme inhibitors, anti-TRAIL, MEK inhibitors, etc.

In specific aspects the HER3-binding molecules of the invention, e.g.,antibodies or antigen-binding fragments thereof, can be administered incombination with antibodies or antibody fragments targeting epidermalgrowth factor receptor (EGFR), e.g., cetuximab (Erbitux®, Imclone),panitumumab (Vectibix®, Amgen), matuzumab/EMD72000 (Merck Serono),MM-151 oligoclonal (Merrimack), nimotuzumab (TheraCIM, InnGeneKalbiotechy), GA201/RG7160 (Roche), Sym004 (Symphogen), MEHD-7945A(EGFR/HER3 dual specific, Genentech). In other specific aspects theHER3-binding molecules of the invention, e.g., antibodies orantigen-binding fragments thereof, can be administered in combinationwith antibodies or antibody fragments targeting HER2, e.g., pertuzumab(rhuMAb 2C4/Omnitarg®, Genentech), trastuzumab (Herceptin®,Genentech/Roche), MM-111 (HER2/HER3 bispecific antibody, Merrimack,e.g., WO 2009/126920). In still other specific aspects the HER3-bindingmolecules of the invention, e.g., antibodies or antigen-bindingfragments thereof, can be administered in combination with antibodies orantibody fragments that also target HER3, e.g., MEHD-7945A/RG7597(EGFR/HER3 dual specific, Genentech, e.g., WO 2010108127), MM-121(Merrimack, e.g., WO 2008/100624), MM-111 (HER2/HER3 bispecificantibody, Merrimack, e.g., WO 2009/126920), AV-203 (Aveo, e.g., WO2011/136911), AMG888 (Amgen, WO 2007/077028), HER3-8 (ImmunogGen, e.g.,WO 2012/019024). In further specific aspects the HER3-binding moleculesof the invention, e.g., antibodies or antigen-binding fragments thereof,can be administered in combination with antibodies or antibody fragmentstargeting HER4. In a specific aspect, the HER3-binding molecules of theinvention can be administered in combination with an antibody thattargets EGFR, or HER2 (e.g., cetuximab or trastuzumab). In a furtherspecific aspect, the HER3-binding molecules of the invention can beadministered in combination with antibody drug conjugates that targetsHER2 (e.g., trastuzumab emtansine, Genentech/Roche). It is contemplatedthat the HER3-binding molecules of the invention enhance theinternalization and degradation of a co-receptor induced by the bindingof an antibody to the co-receptor and will thus, enhance the efficacy ofan antibody and/or antibody drug conjugate that targets EGFR, HER2and/or HER4.

In other aspects, the HER3-binding molecules of the invention can beadministered in combination with tyrosine kinase inhibitors. In someother specific aspects, the HER3-binding molecules of the invention canbe administered in combination with inhibitors of the tyrosine kinaseactivity associated with EGFR and/or HER2/neu, e.g., lapatinib. Inspecific aspects the HER3-binding molecules of the invention, can beadministered in combination with small molecule inhibitors of theepidermal growth factor receptor(s) (e.g., EGFR, HER2, HER4) e.g.,gefitinib (lressa®, Astrazeneca); canertinib/CI-1033 (Pfizer); lapatinib(Tykerb®, GlaxoSmithKline), erlotinib (Tarceva®, OSI Pharma), afatinib(Tovok®/Tomtovok®, Boehringer Ingelheim), neratinib (HKI-272, Pfizer).

In some aspects, the HER3-binding molecules of the invention can beadministered in combination with antimitotic agents. In some specificaspects, the HER3-binding molecules of the invention can be administeredin combination with agents that stabilize the mitotic spindlemicrotubule assembly, e.g, paclitaxel or docetaxel.

In some aspects, the HER3-binding molecules of the invention can beadministered in combination with MEK (mitogen-activated protein kinase(MAPK) kinase, also known as MAPKK) inhibitors, e.g., selumetinib(AZD6244, ARRY-142866, AstraZeneca), WX-554 (Wilex), trametinib(GlaxoSmithKline), refametinib (Ardea Biosciences), E-6201 (Eisai),MEK-162 (Novartis). In a particular aspect, the combination of a MEKinhibitor and a HER3-binding molecule of the invention is moreefficacious than either agent alone. In a specific aspect, aHER3-binding molecule of the invention is administered in combinationwith selumetinib.

Where the combined therapies comprise administration of an anti-HER3binding molecule in combination with administration of anothertherapeutic agent, the methods of the invention encompasscoadministration, using separate formulations or a single pharmaceuticalformulation, and consecutive administration in either order. In someaspects, the anti-HER3 antibodies described herein are administered incombination with other drugs, wherein the antibody or antigen-bindingfragment, variant, or derivative thereof and the therapeutic agent(s)can be administered sequentially, in either order, or simultaneously(i.e., concurrently or within the same time frame).

The combination therapy can provide “synergy” and prove “synergistic”,i.e., the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect can be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect can be attained when the compounds are administered or deliveredsequentially, e.g., by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e., serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

In some aspects, the HER3-binding molecule, e.g., an anti-HER3 antibodyor antigen binding fragment thereof of the invention can be administeredin a synergistic combination with a epidermal growth factor receptor(EGFR) inhibitor. In some aspects, the EGFR inhibitor is an antibody. Inspecific aspects, the EGFR inhibitor antibody is Erbitux® (cetuximab) orpanitumumab (Vectibix®). In specific aspects the HER3-binding moleculesof the invention, e.g., antibodies or antigen-binding fragments thereof,can be administered in a synergistic combination with inhibitors of thetyrosine kinase activity associated with EGFR and/or HER2/neu, e.g.,lapatinib. In some aspects, the HER3-binding molecule, e.g., ananti-HER3 antibody or antigen binding fragment thereof of the inventioncan be administered in a synergistic combination with a HER2 inhibitor.In some aspects, the HER2 inhibitor is an antibody. In specific aspects,the HER2 inhibitor antibody is pertuzumab (rhuMAb 2C4/Omnitarg®,Genentech), trastuzumab (Herceptin®, Genentech/Roche) or trastuzumabemtansine (Genentech/Roche). In specific aspects the HER3-bindingmolecules of the invention, e.g., antibodies or antigen-bindingfragments thereof, can be administered in a synergistic combination withinhibitors of the tyrosine kinase activity associated with HER2/neu,e.g., lapatinib. In some aspects, the HER3-binding molecules of theinvention can be administered in a synergistic combination with anantimitotic agent. In some specific aspects the antimitotic agentstabilizes the mitotic spindle microtubule assembly. In some specificaspects, the antimitotic agent is paclitaxel or docetaxel. In somespecific embodiments, the 2C2 antibody can be administered in asynergistic combination with a growth factor receptor (EGFR) inhibitor.In some specific embodiments, the EGFR inhibitor is an antibody. Inspecific embodiments, the EGFR inhibitor antibody administeredsynergistically with the 2C2 antibody is Erbitux® (cetuximab). Inspecific embodiments the 2C2 antibody can be administered in asynergistic combination with inhibitors of the tyrosine kinase activityassociated with EGFR and/or HER2/neu, e.g., lapatinib. In someembodiments, the 2C2 antibody can be administered in a synergisticcombination with an antimitotic agent. In some specific embodiments, theantimitotic agent administered synergistically with the 2C2 antibodystabilizes the mitotic spindle microtubule assembly. In some specificembodiments, the antimitotic agent administered synergistically with the2C2 antibody is paclitaxel.

In one aspect, the cancer comprises the KRAS mutation. In specificaspects, the KRAS mutation is located at codon 12 of a human KRAS gene.As demonstrated in the Examples section, anti-HER3 antibodies disclosedherein as capable on inhibiting the growth of tumor cells that comprisea KRAS mutation, either when used as a single agent (monotherapy) or incombination with another therapeutic agent.

A further aspect is the use of anti-HER3 binding molecules, e.g.,antibodies or antigen-binding fragments, variants, or derivativesthereof, for diagnostic monitoring of protein levels in tissue as partof a clinical testing procedure, e.g., to determine the efficacy of agiven treatment regimen. For example, detection can be facilitated bycoupling the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

VII. Pharmaceutical Compositions and Administration Methods

Methods of preparing and administering anti-HER3 binding molecules,e.g., antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention to a subject in need thereof are well known toor are readily determined by those skilled in the art. The route ofadministration of the anti-HER3 binding molecule, e.g, antibody, orantigen-binding fragment, variant, or derivative thereof can be, forexample, oral, parenteral, by inhalation or topical. The term parenteralas used herein includes, e.g., intravenous, intraarterial,intraperitoneal, intramuscular, subcutaneous, rectal, or vaginaladministration. However, in other methods compatible with the teachingsherein, anti-HER3 binding molecules, e.g., antibodies, orantigen-binding fragments, variants, or derivatives thereof, of theinvention can be delivered directly to the site of the adverse cellularpopulation thereby increasing the exposure of the diseased tissue to thetherapeutic agent.

As discussed herein, anti-HER3 binding molecules, e.g., antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be administered in a pharmaceutically effective amount forthe in vivo treatment of HER3-expressing cell-mediated diseases such ascertain types of cancers.

The pharmaceutical compositions used in this invention can comprisepharmaceutically acceptable carriers, including, e.g., water, ionexchangers, proteins, buffer substances, and salts. Preservatives andother additives can also be present. The carrier can be a solvent ordispersion medium. Suitable formulations for use in the therapeuticmethods disclosed herein are described in Remington's PharmaceuticalSciences (Mack Publishing Co.) 16th ed. (1980). In some aspects, theHER3-binding molecules of the invention are formulated in a refrigerator(2-8° C.) stable composition. In a particular aspect, the refrigeratorstable composition comprises 25 mM histidine/histidine HCL, 205 mMsucrose, 0.02% polysorbate 80 at pH 6.0. In another particular aspect,the HER3-binding molecules of the invention are formulated at 25-100mg/ml in the refrigerator stable composition.

In any case, sterile injectable solutions can be prepared byincorporating an active compound (e.g., an anti-HER3 antibody, orantigen-binding fragment, variant, or derivative thereof, by itself orin combination with other active agents) in the required amount in anappropriate solvent followed by filtered sterilization. Further, thepreparations can be packaged and sold in the form of a kit. Sucharticles of manufacture can have labels or package inserts indicatingthat the associated compositions are useful for treating a subjectsuffering from, or predisposed to a disease or disorder.

Parenteral formulations can be a single bolus dose, an infusion or aloading bolus dose followed with a maintenance dose. These compositionscan be administered at specific fixed or variable intervals, e.g., oncea day, or on an “as needed” basis.

The composition can be administered as a single dose, multiple doses orover an established period of time in an infusion. Dosage regimens alsocan be adjusted to provide the optimum desired response (e.g., atherapeutic or prophylactic response).

Therapeutically effective doses of the compositions of the presentinvention, for treatment of HER3-expressing cell-mediated diseases suchas certain types of cancers including e.g., colon cancer, lung cancer,gastric cancer, head and neck squamous cells cancer, melanoma,pancreatic cancer, prostate cancer, and breast cancer, vary dependingupon many different factors, including means of administration, targetsite, physiological state of the patient, whether the patient is humanor an animal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human, butnon-human mammals including transgenic mammals can also be treated.Treatment dosages can be titrated using routine methods known to thoseof skill in the art to optimize safety and efficacy.

The amount of at least one anti-HER3 binding molecule, e.g., antibody orbinding fragment, variant, or derivative thereof to be administered isreadily determined by one of ordinary skill in the art without undueexperimentation given the disclosure of the present invention. Factorsinfluencing the mode of administration and the respective amount of atleast one anti-HER3 binding molecule, e.g., antibody, antigen-bindingfragment, variant or derivative thereof include, but are not limited to,the severity of the disease, the history of the disease, and the age,height, weight, health, and physical condition of the individualundergoing therapy. Similarly, the amount of anti-HER3 binding molecule,e.g., antibody, or fragment, variant, or derivative thereof, to beadministered will be dependent upon the mode of administration andwhether the subject will undergo a single dose or multiple doses of thisagent.

The present invention also provides for the use of an anti-HER3 bindingmolecule, e.g., an antibody or antigen-binding fragment, variant, orderivative thereof, in the manufacture of a medicament for treating atype of cancer, including, e.g., colon cancer, lung cancer, gastriccancer, head and neck squamous cells cancer, melanoma, pancreaticcancer, prostate cancer, and breast cancer.

The invention also provides for the use of an anti-HER3 bindingmolecule, e.g., antibody of the invention, or antigen-binding fragment,variant, or derivative thereof, in the manufacture of a medicament fortreating a subject for treating a type of cancer. In certain aspects,the medicament is used in a subject that has been pretreated with atleast one other therapy. By “pretreated” or “pretreatment” is intendedthe subject has received one or more other therapies (e.g., been treatedwith at least one other anti-cancer therapy) prior to receiving themedicament comprising the anti-HER3 binding molecule, e.g., antibody orantigen-binding fragment, variant, or derivative thereof. It is notnecessary that the subject was a responder to pretreatment with theprior therapy or therapies. Thus, the subject that receives themedicament comprising the anti-HER3 binding molecule, e.g., an antibodyor antigen-binding fragment, variant, or derivative thereof could haveresponded, or could have failed to respond to pretreatment with theprior therapy, or to one or more of the prior therapies wherepretreatment comprised multiple therapies.

The invention also provides for the co-administration of an anti-HER3binding molecule, e.g., antibody of the invention, or antigen-bindingfragment, variant, or derivative thereof and at least one other therapy.The anti-HER3 antibody and the at least one other therapy can beco-administered together in a single composition or can beco-administered together at the same time or overlapping times inseparate compositions.

The invention also provides for the use of an anti-HER3 bindingmolecule, e.g., antibody of the invention, or antigen-binding fragment,variant, or derivative thereof, in the manufacture of a medicament fortreating a subject for treating cancer, wherein the anti-HER3 bindingmolecule is administered before a subject has been treated with at leastone other therapy.

VIII. Diagnostics

The invention further provides a diagnostic method useful duringdiagnosis of HER3-expressing cell-mediated diseases such as certaintypes of cancer including, e.g., colon cancer, lung cancer, gastriccancer, head and neck squamous cells cancer, melanoma, pancreaticcancer, prostate cancer, and breast cancer, which involves measuring theexpression level of HER3 protein or transcript in tissue or other cellsor body fluid from an individual and comparing the measured expressionlevel with a standard HER3 expression level in normal tissue or bodyfluid, whereby an increase in the expression level compared to thestandard is indicative of a disorder.

The anti-HER3 antibodies of the invention and antigen-binding fragments,variants, and derivatives thereof, can be used to assay HER3 proteinlevels in a biological sample using classical immunohistological methodsknown to those of skill in the art (e.g., see Jalkanen, et al., J. Cell.Biol. 101:976-985 (1985); Jalkanen et al., J. Cell Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting HER3 proteinexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA), immunoprecipitation, or Western blotting. Suitable assaysare described in more detail elsewhere herein.

By “assaying the expression level of HER3 polypeptide” is intendedqualitatively or quantitatively measuring or estimating the level ofHER3 polypeptide in a first biological sample either directly (e.g., bydetermining or estimating absolute protein level) or relatively (e.g.,by comparing to the disease associated polypeptide level in a secondbiological sample). HER3 polypeptide expression level in the firstbiological sample can be measured or estimated and compared to astandard HER3 polypeptide level, the standard being taken from a secondbiological sample obtained from an individual not having the disorder orbeing determined by averaging levels from a population of individualsnot having the disorder. As will be appreciated in the art, once the“standard” HER3 polypeptide level is known, it can be used repeatedly asa standard for comparison.

The invention further provides a diagnostic method useful duringdiagnosis of HER3-expressing cell-mediated diseases such as certaintypes of cancer including, e.g., colon cancer, lung cancer, gastriccancer, head and neck squamous cells cancer, melanoma, pancreaticcancer, prostate cancer, and breast cancer, which involves measuring theactivity level of HER3 protein in tissue or other cells or body fluidfrom an individual and comparing the measured activity level with astandard HER3 activity level in normal tissue or body fluid, whereby anincrease in the activity level compared to the standard is indicative ofa disorder.

The invention further provides a diagnostic method useful duringtreatment of HER3-expressing cell-mediated diseases such as certaintypes of cancer including, e.g., colon cancer, lung cancer, gastriccancer, head and neck squamous cells cancer, melanoma, pancreaticcancer, prostate cancer, and breast cancer, which involves measuring theactivity level of HER3 protein in tissue or other cells or body fluidfrom an individual during treatment of a HER3-expressing cell-mediateddisease and comparing the measured activity level with a standard HER3activity level in normal tissue or body fluid and/or comparing themeasured activity level with a standard HER3 activity level in tissue orbody fluid obtained from the individual prior to treatment, whereby adecrease in the activity level compared to the standard is indicative ofan inhibition of HER3 activity.

By “assaying the activity level of HER3 protein” is intendedqualitatively or quantitatively measuring or estimating the activity ofHER3 protein in a first biological sample either directly (e.g., bydetermining or estimating absolute activity level) or relatively (e.g.,by comparing to the activity level in a second biological sample). HER3protein activity level in the first biological sample can be measured orestimated and compared to a standard HER3 protein activity, the standardbeing taken from a second biological sample obtained from an individualnot having the disorder or being determined by averaging levels from apopulation of individuals not having the disorder or from an individualprior to treatment. As will be appreciated in the art, once the“standard” HER3 protein activity level is known, it can be usedrepeatedly as a standard for comparison. In certain aspects, theactivity level of HER3 in a biological sample is measured or estimatedor compared by detecting phosphorylated HER3 in a biological sample. Ina specific aspect, the activity level of HER3 in a biological sample ismeasured or estimated or compared by detecting phosphorylated HER3 in askin biopsy, wherein the skin is stimulated with HRG prior to or afterbiopsy.

By “biological sample” is intended any biological sample obtained froman individual, cell line, tissue culture, or other source of cellspotentially expressing HER3. Methods for obtaining tissue biopsies andbody fluids from mammals are well known in the art.

In some aspects, the bioactivity of a HER3 inhibitor (e.g., anti-HER3antibody of the invention and antigen-binding fragments, variants andderivatives thereof) administered to a subject can be detected using anex-vivo assay. In particular aspects the ex-vivo assay comprisesdetecting the level of phosphorylated HER3 in a skin biopsy, wherein theskin is stimulated with HRG prior to or after biopsy. In a specificaspect matched skin biopsies are taken from a subject that has beenadministered the HER3 inhibitor. In a specific aspect, HRG is injectedunder a first area of the skin and a control buffer is injected under asecond area of the skin of a subject administered the HER3 inhibitor,wherein after a desired amount of time (e.g., 10-60 minutes) a biopsy istaken from the first and second areas of the skin. In an alternativeaspect, a first skin biopsy is treated with HRG and a second skin biopsyis treated with a control buffer, wherein the first and the secondbiopsies are matched skin biopsies taken from a subject that has beenadministered the HER3 inhibitor. In another specific aspect, the levelof phosphorylated HER3 is detected in the skin biopsies. In certainaspects, the difference in the level of phosphorylated HER3 between thefirst (HRG treated) and the second (control buffer treated) biopsy isdetermined. In certain aspects, the skin biopsy is homogenized and thelevel of phosphorylated HER3 is detected by ELISA. In still otheraspects, the levels of phosphorylated HER3 in the skin biopsies from asubject that has been administered the HER3 inhibitor is compared to thelevels of phosphorylated HER3 in skin biopsies from a control subjectthat has not been administered the HER3 inhibitor, wherein a reductionin the level of phosphorylated HER3 in the skin biopsies of the subjectthat has been administered the HER3 inhibitor is a measure of thebioactivity of the HER3 inhibitor. In alternative aspects, the levels ofphosphorylated HER3 in the skin biopsies from a subject that has beenadministered the HER3 inhibitor is compared to the levels ofphosphorylated HER3 in skin biopsies from the same subject taken priorto the administration of the HER3 inhibitor, wherein a reduction in thelevel of phosphorylated HER3 in the skin biopsies of the subject afteradministration of the HER3 inhibitor is a measure of bioactivity of theHER3 inhibitor. Other specific aspects of the methods are detailed inthe Examples section 5.15.

IX. Kits Comprising HER3-Binding Molecules

The present invention provides kits that comprise the HER3-bindingmolecule, e.g., an anti-HER3 antibody or antigen binding fragmentthereof of the invention described herein and that can be used toperform the methods described herein. In certain aspects, a kitcomprises at least one purified anti-HER3 antibody or an antigen-bindingfragment thereof in one or more containers. In some aspects, the kitscontain all of the components necessary and/or sufficient to perform adetection assay, including all controls, directions for performingassays, and any necessary software for analysis and presentation ofresults. One skilled in the art will readily recognize that thedisclosed HER3-binding molecule, e.g., an anti-HER3 antibody or antigenbinding fragment thereof of the present invention can be readilyincorporated into one of the established kit formats which are wellknown in the art.

X. Immunoassays

Anti-HER3 binding molecules, e.g., antibodies or antigen-bindingfragments thereof, variants, or derivatives thereof of the molecules ofthe invention can be assayed for immunospecific binding by any methodknown in the art. The immunoassays that can be used include but are notlimited to competitive and non-competitive assay systems usingtechniques such as Western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al., eds,(1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc.,NY) Vol. 1, which is incorporated by reference herein in its entirety).

HER3-binding molecules, e.g., anti-HER3 antibodies or antigen-bindingfragments thereof, variants, or derivatives thereof of the molecules ofthe invention, can be employed histologically, as in immunofluorescence,immunoelectron microscopy or non-immunological assays, for in situdetection of HER3 receptors or conserved variants or peptide fragmentsthereof. In situ detection can be accomplished by removing ahistological specimen from a patient, and applying thereto a labeledHER3-binding molecule, e.g., an anti-HER3 antibody or antigen-bindingfragment thereof, variant, or derivative thereof, preferably applied byoverlaying the labeled HER3-binding molecule (e.g., and antibody orfragment) onto a biological sample. Through the use of such a procedure,it is possible to determine not only the presence of HER3, or conservedvariants or peptide fragments, but also its distribution in the examinedtissue. Using the present invention, those of ordinary skill willreadily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

The binding activity of a given lot of HER3-binding molecule, e.g.,anti-HER3 antibody or antigen-binding fragment thereof, variant, orderivative thereof can be determined according to well-known methods.Those skilled in the art will be able to determine operative and optimalassay conditions for each determination by employing routineexperimentation.

Methods and reagents suitable for determination of bindingcharacteristics of an isolated HER3-binding molecule, e.g., anti-HER3antibody or antigen-binding fragment thereof, variant, or analtered/mutant derivative thereof, are known in the art and/or arecommercially available. Equipment and software designed for such kineticanalyses are commercially available (e.g., BIAcore, BIAevaluationsoftware, GE Healthcare; KinExa Software, Sapidyne Instruments).

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, Sambrook etal., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; ColdSpring Harbor Laboratory Press); Sambrook et al., ed. (1992) MolecularCloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D.N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984)Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hamesand Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins,eds. (1984) Transcription And Translation; Freshney (1987) Culture OfAnimal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRLPress) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; thetreatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller andCalos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (ColdSpring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols.154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods InCell And Molecular Biology (Academic Press, London); Weir and Blackwell,eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al. (1989) CurrentProtocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

General principles of antibody engineering are set forth in Borrebaeck,ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). Generalprinciples of protein engineering are set forth in Rickwood et al., eds.(1995) Protein Engineering, A Practical Approach (IRL Press at OxfordUniv. Press, Oxford, Eng.). General principles of antibodies andantibody-hapten binding are set forth in: Nisonoff (1984) MolecularImmunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward(1984) Antibodies, Their Structure and Function (Chapman and Hall, NewYork, N.Y.). Additionally, standard methods in immunology known in theart and not specifically described are generally followed as in CurrentProtocols in Immunology, John Wiley & Sons, New York; Stites et al.,eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange,Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods inCellular Immunology (W.H. Freeman and Co., NY).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination(John Wiley & Sons, NY); Kennett et al., eds. (1980) MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses (PlenumPress, NY); Campbell (1984) “Monoclonal Antibody Technology” inLaboratory Techniques in Biochemistry and Molecular Biology, ed. Burdenet al., (Elsevere, Amsterdam); Goldsby et al., eds. (2000) KubyImmunnology (4th ed.; H. Freemand & Co.); Roitt et al. (2001) Immunology(6th ed.; London: Mosby); Abbas et al. (2005) Cellular and MolecularImmunology (5th ed.; Elsevier Health Sciences Division); Kontermann andDubel (2001) Antibody Engineering (Springer Verlan); Sambrook andRussell (2001) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Press); Lewin (2003) Genes VIII (Prentice Hall 2003); Harlow andLane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press);Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

Aspects of the present disclosure can be further defined by reference tothe following non-limiting examples, which describe in detailpreparation of certain antibodies of the present disclosure and methodsfor using antibodies of the present disclosure. It will be apparent tothose skilled in the art that many modifications, both to materials andmethods, can be practiced without departing from the scope of thepresent disclosure.

Example 1 Methods for Isolation/Optimization of anti-HER3 MonoclonalAntibodies

1.1. Antigens and Cell Lines

Recombinant human Herl(ECD)/Fc chimera, human HER2(ECD)/Fc chimera,human HER3(ECD)/Fc chimera and human Her4(ECD)/Fc were all purchasedfrom R&D Systems (Minneapolis, Minn.) and were fused to the C-terminal6× Histidine-tag via a linker peptide. Recombinant mouse HER3(ECD)/Fcchimera was generated in house. Human KPL-4 breast cancer cells werecultured in DMEM supplemented with 5% fetal bovine serum (FBS).

1.2. Library Selection of HER3 Binders—Identification of Clone 16Antibody (CL16)

The unlabeled and biotinylated HER3(ECD)/Fc were used as the targets forselection of HER3 binders from Dyax's Fab 310 human Fab phage displaylibrary (Dyax, Cambridge, Mass.). Two arms of panning were carried out:captured panning and in solution panning. For the captured panning,input phage were first incubated with polyclonal human IgG captured onimmunotubes via immobilized recombinant Protein A/G, and then selectedwith unlabeled target captured on immunotubes via immobilizedrecombinant Protein A/G.

In the in solution panning, input phage were allowed to incubate withpolyclonal human IgG, streptavidin-coated magnetic beads with quenchedbiotin for deselection and then selected with biotinylated target withsubsequent incubation with streptavidin-coated magnetic beads to capturephage bound to the target. After removal of unbound phage by washingextensively with TPBS (1×PBS/0.1% Tween-20), the bound phage were elutedwith 100 mM TEA (triethylamine). Eluted phage and the remaining phage onbeads from in solution panning were subsequently amplified, andsubjected to further rounds of selection. Three rounds of selection werecarried out for each arm of selection.

The percentage of positive binding phages ranged from less than 1% usingcapture panning up to 68% using three rounds of in solution panning(TABLE 3).

TABLE 3 Screening of HER3 binders. Captured Second Round In Third RoundIn Panning Panning Solution Panning Solution Panning Total clonesscreened 380 285 475 Positive clones 1 7 322 Positive rate (%) <1 2 68

1.3. Screening for Human and Mouse HER3 Binders by Phage ELISA

Phage enriched from the second and the third rounds of selection werescreened by phage ELISA for human and mouse HER3 binding. 96-well halfarea plates were coated with 5 μg/ml, 50 μl per well of differentantigens diluted in 1× PBS, pH 7.4 overnight at 4° C. The coated plateswere blocked with 3% (w/v) non-fat milk in TPBS for 1 hour at roomtemperature, and washed two times with TPBS. The plates were thenincubated for 1 h with overnight phage supernatant. After washing tentimes with TPBS, the plates were incubated with horseradish peroxidase(HRP)-conjugated anti-M13 antibody for 1 hour, and washed ten times.Plates were developed with tetramethylbenzidine (TMB) peroxidasesubstrate solution, the reactions were stopped with 0.18M of H₂SO₄, andplates were read at 450 nm on an ELISA plate reader.

29 unique positive binders were identified that were cross reactive tomurine HER3 (as a HER3-Fc fusion). None of the identified binders showedcross reactivity to HER2 or Her4 (data not shown).

1.4. Reformatting of Fabs into Whole IgG Antibodies and Expression

The immunoglobulin variable light chain (VL) and variable heavy chain(VH) from positive phage clones were generated by PCR and inserted intoa human IgG1 expression vector containing the lambda light chainconstant region and the CH1-hinge-CH2-CH3 IgG1 region. To express IgG1antibodies, human embryonic kidney 293-F cells were transientlytransfected with the reformatted IgG vectors using 293fectin™ reagent(Invitrogen, Carlsbad, Calif.). Conditioned media were harvested 10 daysafter transfection, pooled, and sterile-filtered. IgGls were purifiedusing protein A beads. The final eluted IgG1s were dialyzed against PBS,and IgG1 concentrations were determined by protein quantitation assay.

Clone 16 (CL16; SEQ ID NOs: 1 and 2, VL and VH amino acid sequences,respectively) was reformatted to human IgG1

1.5. Determination of Internalization of Clone 16 Antibody (CL16) byImmunofluorescence

Human breast cancer KPL-4 cells were labeled with Clone 16 antibody(CL16). Incubation of the cells with CL16 lead to an increase in HER3endocytosis, which prevented the receptor from forming active signalingcomplexes with HER2 at the cell surface.

Cell surface attached CL16 antibodies were allowed to internalize byincubating the cells under growth conditions for either zero(non-internalized) or 2.5 hours (internalized) (FIG. 1). All cells werethen fixed with 3.7% paraformaldehyde, washed in PBS, permeabilized with0.5% Triton X-100 in PBS, and stained with 1 μg/ml Alexa Fluor® 488 goatanti-human IgG (Invitrogen) prior to addition of antifade mounting mediaand fluorescent microscopy examination. The CL16 antibody was found tointernalize in KPL-4 cells. At time zero KPL-4 cells showed intense cellsurface staining (FIG. 1, 0 hours, top panel), after incubation undergrowth conditions for 2.5 hours the cell surface staining was diminishedand replaced by intracellular punctuate staining indicative ofinternalization (FIG. 1, 2.5 hours, bottom panels).

1.6. Construction of a Phage Vector Expressing Clone 16 Fab

DNA encoding the antigen binding fragment (Fab) of the antibody Clone 16was cloned into a modified, M13-based phage expression vector previouslydescribed by Dall'Acqua et al. (Dall'Acqua et al., 2005, Methods.36:43-60). In this modified vector, a human lambda (λ) constant regionDNA was engineered in place of the human kappa (κ) light chain. Theexpression of Fab fragment is under the control of the LacZ promoter andsecretion of the Fab fragment is enabled by the phage P3 signalsequences fused to the N-termini of either the VH and the VL chains ofthe Fab fragment. The cloning was carried out by hybridizationmutagenesis as described by Kunkel (Kunkel, T. A., 1985, Proc. Natl.Acad. Sci. USA; 82:488-492) and Wu (Wu, H., 2003, Methods Mol. Biol.207:197-212).

Briefly, the variable regions of clone 16 IgG were amplified bypolymerase chain reaction (PCR). By hybridization followed by DNApolymerization reaction, the clone 16 variable light region wasintegrated in frame with the human lambda constant region, and thevariable heavy region was cloned in frame with the human heavy chainconstant region 1 (CH1), respectively. The phage vector containing theClone 16 Fab fragment was then grown in Escherichia coli CJ236 strain toproduce uridine (U) containing single stranded DNA (ssDNA) as describedby Wu and An (Wu, H. and An, L L., 2003, Methods Mol. Biol. 207:213-33).The uridine containing ssDNA was used as the template to introducedesigned mutations for improving binding affinity to HER3.

1.7. Germlining of Clone 16 (CL16)

Sequence analysis shows that the VH frameworks of Clone 16 (CL16) shares100% sequence identity with VH germline gene 3-23 while VL frameworksdiffer at 6 positions from its closest germline gene 47*01. Site directmutagenesis to change each and all of the amino acids that differ fromthe germline gene 47*01 was performed. Specifically, six point mutationswere introduced into the light chain variable regions as follows: Y2S,E3V, S18R, M211, H38Q and S50Y where the first letter represents the oneletter amino acid code of the original Clone 16, the number representsthe framework residue number (as per Kabat), and the second letterrepresents the one letter amino acid code of the germline sequence. Seesequences in FIG. 2A and FIG. 2C, corresponding to the original VL CL16and germlined (GL) VL CL16, respectively. The resultant variants wereexpressed as Fab and their binding to the recombinant HER3 protein wasdetermined by ELISA.

The binding results showed that the H38Q amino acid mutation inframework 2 improved binding over the parental Clone 16 as measured byELISA. In contrast, the S49Y mutation in the same framework had negativeimpact on binding. Other point mutations showed no impact on HER3binding. The fully germlined mutant with all 6 non-germline amino acidsmutated showed a similar degree of reduced binding as the 550Y pointmutation, indicating that amino acid S50 participates in binding.Further testing of the clone with all the germline point mutationsexcept 550Y retained and/or increased binding to HER3 comparing to theparental clone 16. This partially germlined clone, Clone 16 (GL) (alsoreferred to here as “GL-P6”), was used as the template for furtheraffinity optimization.

1.8. Affinity Optimization of Clone 16 (CL16)

Each amino acid of all 6 complementary-determining regions (CDRs) ofgermlined clone GL-P6 was individually mutated to other 20 amino acidsusing a hybridization mutagenesis method (Kunkel, 1985). Two sets of DNAprimers, one containing a NSS codon encoding 8 amino acids and the othercontaining a NWS codon encoding 12 different amino acids, were used tointroduce mutations to each targeted CDR position. The individualdegenerate primers were used in hybridization mutagenesis reactions.Briefly, each degenerate primer was phosphorylated, then used in a 10:1ratio with the uridinylated GL-16 Fab ssDNA. The mixture was heated to95° C. then cooled down to 55° C. over 1 hour. Thereafter, T4 ligase andT7 DNA polymerase were added and the mix was incubated for 1.5 hours at37° C. Synthesis products for VH and VL CDRs were pooled respectively;however, NSS and NWS libraries were kept separate and screenedindependently. Typically, 1 μL of the pooled library DNA waselectroporated into XL1-Blue for plaque formation on XL1-Blue bacteriallawn or for production of Fab fragments (Wu and An, 2003).

1.9. Primary Screening of the Fab Library

The primary screen consisted of a single point ELISA (SPE) assay whichwas carried out using culture supernatant of bacteria grown in 96-wellplates (deep well) and infected with individual recombinant M13 clonesas described elsewhere (Wu and An, 2003). Briefly, this capture ELISAinvolved coating individual wells of a 96-well Maxisorp Immunoplate withapproximately 50 ng of a sheep anti-human Fd antibody (BiodesignInternational, ME) in a carbonate buffer at pH 8.5 overnight at 4° C.The next day, the plate was blocked with 3% BSA in PBS buffer for 1 h atroom temperature. Fab supernatant was then added to the plate andincubated at room temperature for 1 hr. After washing, 0.1 μg ofbiotinylated HER3 protein was added to the well and the mixture wasincubated for 1.5 h at room temperature. This was followed by incubationwith neutravidin-horseradish peroxydase (HRP) conjugate (Pierce, Ill.)for approximately 40 min at room temperature. HRP activity was detectedwith tetra-methyl-benzidine (TMB) substrate and the reaction quenchedwith 0.2 M H₂SO₄. Plates were read at 450 nm.

Clones exhibiting an optical density (OD) signal at 450 nm greater thanthe parental clone GL-P6 Fab were picked and regrown (15 mL) (Wu and An,2003) and re-assayed by ELISA (as described above) in duplicate toconfirm positive results. Clones that repeatedly exhibited a signalgreater than that of the GL-P6 Fab were sequenced. The Fab proteinconcentration of each clone that had a CDR change was then determined bya quantitative Fab ELISA, where a Fab with known concentration was usedas a reference. The Fab concentration was determined by comparing theELISA signals with the signals generated by the reference Fab. Thebinding assay was repeated once more for all positive variants undernormalized Fab concentrations in order to determine the relative bindingaffinity of the mutant Fabs and the parental GL-P6 Fab.

The binding ELISA showed that two VH variants, designated clone 14C7 andclone 15D12, which contained the Y501 or Y5OV point mutations,respectively, in CDR2 displayed approximately a 5-fold improvement inHER3 binding over the parental, germlined clone GL-P6. In the VLmutagenesis campaign, several single mutations either in CDR1, e.g.,clone 4H6 (comprising the S24R point mutation), clone 6E3 (comprisingthe S27L point mutation) or in CDR3, e.g., clone 5H6 (comprising theS94G point mutation), clone 8A3 (comprising the S96aI point mutation),clone 4C4 (comprising the S96aR point mutation), clone 2B11 (comprisingthe S96aP point mutation) and clone 2D1 (comprising the V97A mutation)displaying improved binding were identified.

Most notably, the substitution of amino acid S96a of VL-CDR3 with eitherIsoleucine (I), Arginine (R) or Proline (P) resulted in a 3.5-fold,8.6-fold and 32-fold binding improvement, respectively.

1.10. Combinatorial Screening of the Fab Library

The point mutations in VH and VL determined to be beneficial for bindingto HER3 were further combined to gain additional binding synergy. Thecombinatorial mutants were expressed as Fab and screened using the HER3binding ELISA. While combining either one of the Y50I or Y50V pointmutation in the VH chain of the Fab fragment with the VL mutationsappeared to have no beneficial but reduced binding to HER3, combiningseveral VL mutations further improved binding. These combination of VLmutations include the combinations in clone 1A4 (comprising the L96P,S97P and V100A point mutations), clone 2C2 (comprising the S26L, L96P,S97P and V100A point mutations), clone 2F10 (comprising the S97P andV100A mutations) and clone 3E1 (comprising the S23R, L96P, S97P andV100A point mutations).

1.11. Conversion of the Affinity-optimized Fab Variants to IgG Formatand Antibody Expression of

Singe mutant and combination mutant variants displaying improved bindingwere converted into IgG format for further characterization. Thevariable regions of each variant were amplified by PCR using primersthat encoded restriction sites to facilitate cloning into an IgGmammalian expression vector for expression using HEK 293F cells. Thesecreted, soluble human IgG1 proteins were purified from the conditionedmedia directly on 1 mL HiTrap protein A columns (GE Healthcare, NJ)according to the manufacturer's instructions. Purified human IgG1samples (typically >95% homogeneity, as judged by sodium dodecylsulphate-polyacrylamine gel electrophoresis) were dialyzed against PBS,flash frozen, and stored at −70° C.

Binding of the purified IgGs was examined using a HER3 binding ELISA.The combination mutant IgGs showed improved binding as determined by thetotal binding signal, with 2C2 showing the most significant bindingimprovement over the parental Clone 16 and other combination mutantvariants. Binding of the IgGs to murine and cynomolgus HER3 were alsotested by ELISA. The results showed improved binding of the combinationmutants to these paralogous HER3 species.

Alignment of the amino acid sequences of the light and heavy chainvariable regions for each of the identified single mutations is shown inFIG. 2A and FIG. 2B, respectively. TABLE 4 provides the SEQ ID NOs foreach clone. An alignment of the light chain variable regions for each ofthe combination clones is provided in FIG. 2C.

TABLE 4 SEQ SEQ ID DESCRIPTION ID DESCRIPTION 17 Clone 16 V_(L) aa 21Clone 4H6 V_(L) CDR2 aa 1 Clone 16-germlined V_(L) aa 22 Clone 4H6 V_(L)CDR3 aa 2 Clone 16 V_(H) aa 7 Clone 6E.3 V_(L) aa 18 Clone 16 V_(L) CDR1aa 19 Clone 6E.3 V_(L) CDR1 aa 21 Clone 16 V_(L) CDR2 aa 21 Clone 6E.3V_(L) CDR2 aa 22 Clone 16 V_(L) CDR3 aa 22 Clone 6E.3 V_(L) CDR3 aa 31Clone 16 V_(H) CDR1 aa 9 Clone 2D1 V_(L) aa 32 Clone 16 V_(H) CDR2 aa 18Clone 2D1 V_(L) CDR1 aa 35 Clone 16 V_(H) CDR3 aa 21 Clone 2D1 V_(L)CDR2 aa 8 Clone 2B11 V_(L) aa 28 Clone 2D1 V_(L) CDR3 aa 18 Clone 2B11V_(L) CDR1 aa 10 Clone 3A6 V_(L) aa 21 Clone 2B11 V_(L) CDR2 aa 18 Clone3A6 V_(L) CDR1 aa 25 Clone 2B11 V_(L) CDR3 aa 21 Clone 3A6 V_(L) CDR2 aa14 Clone 1A4 V_(L) aa 29 Clone 3A6 V_(L) CDR3 aa 18 Clone 1A4 V_(L) CDR1aa 11 Clone 4C4 V_(L) aa 21 Clone 1A4 V_(L) CDR2 aa 18 Clone 4C4 V_(L)CDR1 aa 22 Clone 1A4 V_(L) CDR3 aa 21 Clone 4C4 V_(L) CDR2 aa 3 Clone2C2 V_(L) aa 30 Clone 4C4 V_(L) CDR3 aa 19 Clone 2C2 V_(L) CDR1 aa 12Clone 15D12.1 V_(H) aa 21 Clone 2C2 V_(L) CDR2 aa 31 Clone 15D12.1 V_(H)CDR1 aa 23 Clone 2C2 V_(L) CDR3 aa 33 Clone 15D12.1 V_(H) CDR2 aa 16Clone 2F10 V_(L) aa 35 Clone 15D12.1 V_(H) CDR3 aa 18 Clone 2F10 V_(L)CDR1 aa 13 Clone 15D12.2 V_(H) aa 21 Clone 2F10 V_(L) CDR2 aa 31 Clone15D12.2 V_(H) CDR1 aa 24 Clone 2F10 V_(L) CDR3 aa 34 Clone 15D12.2 V_(H)CDR2 aa 15 Clone 3E.1 V_(L) aa 35 Clone 15D12.2 V_(H) CDR3 aa 20 Clone3E.1 V_(L) CDR1 aa 36 V_(H) FW1 aa 21 Clone 3E.1 V_(L) CDR2 aa 37 V_(H)FW2 aa 23 Clone 3E.1 V_(L) CDR3 aa 38 V_(H) FW3 aa 4 Clone 5H6 V_(L) aa39 V_(H) FW4 aa 18 Clone 5H6 V_(L) CDR1 aa 40 V_(L) FW1 germlined aa 21Clone 5H6 V_(L) CDR2 aa 41 V_(L) FW2 aa 26 Clone 5H6 V_(L) CDR3 aa 42V_(L) FW3 aa 5 Clone 8A3 V_(L) aa 43 V_(L) FW4 aa 18 Clone 8A3 V_(L)CDR1 aa 44 V_(L) FW1 original aa 21 Clone 8A3 V_(L) CDR2 aa 45 IgG1constant region* 27 Clone 8A3 V_(L) CDR3 aa 46 IgG1 constant region*—YTE6 Clone 4H6 V_(L) aa 47 Clone 16 V_(L) nt 20 Clone 4H6 V_(L) CDR1 aa 48Clone 16 V_(H) nt * allotype differences are provided V_(L) aaconsensus: [FW₁]X₁GSX₂SNIGLNYVS(SEQ ID NO: 49)[FW₂]RNNQRPS (SEQ ID NO:21)[FW₃]AAWDDX₃X₄X₅GEX₆(SEQ ID NO: 50)[FW₄] wherein [FW₁], [FW₂], [FW₃]and [FW₄] represent VL framework regions, wherein (a) X₁ representsamino acid residues Arginine (R) or Serine (S), (b) X₂ represents aminoacid residues Serine (S) or Leucine (L), (c) X₃ represents amino acidresidues Serine (S) or Glycine (G), (d) X₄ represents amino acidresidues Leucine (L) or Proline (P), (e) X₅ represents amino acidresidues Arginine (R), Isoleucine (I), Proline (P) or Serine (S), and(f) X₆ represents amino acid residues Valine (V) or Alanine (A). V_(H)aa consensus: [FW₅]YYYMQ(SEQ ID NO: 31)[FW₆]X₇IGSSGGVTNYADSVKG (SEQ IDNO: 51)[FW₇]VGLGDAFDI(SEQ ID NO: 35)[FW₈] wherein [FW₅], [FW₆], [FW₇]and [FW₈] represent VH framework regions, wherein X₇ represents aminoacid residues Tyrosine (Y), Isoleucine (I) or Valine (V)

1.12. Anti-HER3 Monoclonal Antibody Binding Studies

The kinetic rate (k_(on), k_(off)) and equilibrium dissociationconstants (K_(D)) for the binding of the anti-HER3 IgGs to theextracellular domain of human HER3 protein were determined usingBIAcore™ surface plasmon resonance technology by measuring the bindingof human HER3 extracellular domain(hu HER3(ECD)) to IgG captured onto asensor chip surface. Individual association (k_(on)) and dissociation(k_(off)) rate constants were then calculated from the resulting bindingcurves using the BIAevaluation software available through the vendor.Data were fit to a 1:1 binding model, which included a term to correctfor mass transport limited binding, should it be detected. From theserate constants, the apparent dissociation binding constant (K_(D)) forthe interaction of IgG with the human HER3 extracellular domain proteinis then calculated from the quotient of k_(off)/k_(on).

From high-resolution BIAcore plots, the association and dissociationrate constants for the binding parental IgG, Clone16, to human HER3extracellular domain were 5.29×10⁵/Ms and 73.0×10⁻⁴/s, respectively,yielding an apparent K_(D) of 14 nM. In comparison, the association rateconstants for the binding of the affinity-improved IgG variants to humanHER3 extracellular domain were similar to those measured for theparental IgG, ranging from 3.41×10⁵ to 4.32×10⁵/Ms. These same plotswere also used to determine the corresponding dissociation rateconstants for the Clone 16 variants, which ranged from 1.60×10⁻⁴ to6.21×10⁻⁴/s. The apparent K_(D)s for the Clone 16 variants werecalculated as described above, and ranged from 0.429 nM (2C2 clonevariant) to 1.44 nM. (P2B11 clone variant). Individual errors for k_(on)and k_(off) were low (≦˜2% of the calculated parameter), and the overallfits to the kinetic data indicated that the use of the 1:1 interactionmodel was appropriate. Also, the evaluation did not indicate the bindingwas mass transport-limited.

TABLE 5 summarizes the biophysical attributes of the combinationmonoclonal clones provided in FIG. 2C, including K_(on), K_(off) andK_(D) values, as well as expression levels and yields.

The 2C2 monoclonal antibody, comprising the 2C2 VL (SEQ ID NO: 3) andthe original C16 VH (SEQ ID NO: 2) was the most affinity-improved leadwith a K_(D) of 0.4 nM, representing a 32-fold improvement from theparental Clone 16 monoclonal antibody. The K_(D) improvement was mostlya result of decreased off-rate. The expression level and productionyields were also assessed. All of the monoclonal antibody clones werewell expressed in a 5 day transient transfection study, with the 2C2monoclonal antibody showing the highest level of expression in thisstudy. All affinity optimized leads showed different extents of affinityimprovement but the 1A4 antibody dropped out due to lower expressionefficiency.

TABLE 5 Summary of biophysical properties of the variousaffinity-optimized leads in comparison with the parental CL16 (Clone 16)antibody. Biacore Biacore Expression Kon KD (nM, KD (nM, Level on YieldClone Calculated (1/Ms) Koff (1/s) IgG Her3 Day 5 (mg/volume, name pI(xE + 5) (xE − 4) down) down) (transient) ml) P2B11 8.21 4.32 6.21 1.53(9x) 0.74 (2.4x) 159 ug/ml 70/500 1A4 8.2 3.41 2.86 0.838 (17x) 0.493(3.6x)  60 ug/ml  53/1200 2C2 8.2 3.73 1.60 0.434 (32x) 0.093 (19x) 148ug/ml 71/600 2F10 8.2 3.54 2.90 0.818 (17x) 0.326 (5x) 130 ug/ml 66/6003E 1 8.32 3.43 1.78 0.52 (26x) 0.286 (6.2x) 125 ug/ml 59/600 Clone 167.83 5.29 73.0 14 1.77 ND ND Note: Each affinity-optimized leadcomprises the clone name VL chain and the original C16 VH

Various cell-based assays were performed to assess the functionalimprovement of the various affinity optimized leads over clone 16 acrossligand-independent (human breast cancer cell line BT-474, ATCC No.HTB-20™) as well as ligand-dependent (human breast cancer cell lineT-47D, ATCC No. HTB-133) models (both cell lines obtained from ATCC),including inhibition of HER3 signaling pathway (pHER3 and pAKT),suppression of cell growth (short-term 6-day growth assay and long-termclonogenic assay), and abrogation of HRG-induced pHER3 in T-47D cells(T-47 differentiated epithelial substrain).

Clonogenic assays were performed as follows. BT-474 cells were plated ata density of 1,000 cells/well into 6-well plates. After overnightattachment, cells were treated with isotype control IgG or the indicatedHER3 monoclonal antibodies following a concentration dose curve. Themedium with the proper doses of monoclonal antibodies was refreshed oncea week for three weeks. At the end of day 21, cells were processed forCell-titer-Glo (CTG) assay to assess the inhibition of colony formationby the various monoclonal antibodies (using control IgG as base-line).IC₅₀ values were derived from Prizm analysis.

The BT-474 6-day growth assay was performed essentially as used for FIG.4 (see Section 2.2 in Example 2, infra). The BT-474 pAKT assay wasperformed essentially as used for FIG. 10 (see Section 2.6.1 in Example2, infra). The T-47D HRG inducible pHER3 assay was performed essentiallyas used for FIG. 3 (see Section 2.1 in Example 2, infra), and the T-47DFACS binding and internalization assay was performed using the sameprotocol used for FIG. 16A (see Section 3.3.1 in Example 3, infra).

The IC₅₀ values and maximal inhibition levels were compiled forcomparison purposes. As shown in TABLE 6, the affinity improved leadsdisplayed a consistent 2-3-fold increased potency across most of theassays. The parental Clone 16 and/or a representative optimized clone,e.g., Clone 2C2 antibody (also referred to simply as 2C2, or 2C2monoclonal antibody) were further characterized in a number of in vitroand in vivo assays as described below.

In addition, mutations were introduced into the Fc region of theoptimized clone 2C2 to extend half-life. Specifically, M252Y, S254T,T256E, numbered according to the EU index as in Kabat. Thishalf-life-optimized molecule is referred to as 2C2-YTE. It will beunderstood that other mutations could be introduced instead of, or incombination with these three, see, e.g., U.S. Pat. No. 7,083,784;International Appl. Pub. No. WO2009058492; Dall'Acqua et al., 2002 J.Immunol. 169:5171-80; Zalevsky et al., 2010, Nature Biotech. 28:157-9).2C2-YTE was show to inhibit BT-474 cell proliferation and colony growthto the same extent as 2C2 (data not shown).

A refrigerator (2-8° C.) stable composition was obtained by formulatingthe antibodies (e.g., 50 mg/ml) in 25 mM histidine/histine HCL, 205 mMsucrose, 0.02% polysorbate 80 at pH 6.0.

TABLE 6 Summary of the biological properties of the affinity optimizedleads in comparison with parental CL16 monoclonal antibody. BT474 BT4746-day BT474 pAKT T47D HRG clonogenic growth assay inducible T47D FACSasssay Inflection Inflection pHer3 binding Clone IC50 % Max point % Maxpoint % Max IC50 % Max Max name (pM) inhibition (pM) inhibition (pM)inhibition (pM) inhibition Kd (pM) GMFI P2B11 26.9 87.1 98 47.5 23.6 6279.8 85 199 1441 1A4 30.7 81.3 133.3 54.5 28.5 62 133 84 281 1577 2C231.9 87.2 62.7 48.3 42.6 61 130.3 85 316 1583 2F10 31.2 80.4 66.7 4946.4 62 127.2 86 306 1527 3E 1 20.8 79.2 85.3 48.1 26.2 66 59.2 86 4471644 Clone 16- 64.5 79.8 280 46 73.1 64 104.4 75 112 1055 PA

Example 2 Characterization of Anti-HER3 Monoclonal Antibodies

2.1. HRG-induced HER3 Phosphorylation (pHER3) Assay in MCF-7 Cells

MCF-7 (ATCC No. HTB-22™) is a human breast cancer cell line with HER3expression but no endogenous HRG expression. MCF-7 cells were plated ata density of 30,000 cells/well in a 96-well plate and were allowed toattach overnight. The cells were then serum-starved for 24 hours beforetreatment. Following serum-starvation, media was removed and replacedwith serum-free media containing test and control antibodies, and thecells incubated at 37° C. for 1 hour. Test antibodies used in thisexample, and in the additional examples provided below, include theanti-HER3 antibodies provided herein such as, Clone 16, 2C2, 2C2-YTE;and anti-HER3 antibodies known in the art, in particular U1-59(International Patent Publication WO 2007077028) and Ab#6 (PatentPublication WO 2008/100624) designated herein as AMG and MM,respectively. Meanwhile, heregulin (HRGβ1, R&D Systems, Minneapolis,Minn.) stock was prepared at 4× (80 ng/ml) in serum-free growth media.At the end of the 1 hour incubation period, HRGβ1 was spiked into wells(20 ng/ml final concentration) and incubated at 37° C. for 20 minutes.At the end of treatment, media was removed and cells were washed withPBS. Cells were lysed in 80 μl Triton X lysis buffer (BostonBioproducts, Ashland, Mass.) with protease and phosphatase inhibitors(Calbiochem, La Jolla, Calif.) and were stored at −20° C. untilanalysis. pHER3 ELISA was then performed following manufacturer'sprotocol (R&D Systems, DYC1769) using half-volume 96-well Corning®Costar® 3690 ELISA plates (Corning Life Science, Lowell, Mass.) and 50μl of cell lysate per well.

HER3 activation, reflected by HER3 phosphorylation (abbreviated aspHER3), was stimulated by cells treatment with HRGβ1. Pre-treatment withanti-HER3 2C2 mAb caused a dose-dependent suppression of the pHER3signal in the pHER3 ELISA assay (FIG. 3, top). The published anti-HER3monoclonal antibodies MM and AMG were also active in this assay,however, 2C2 was approximately 5-fold more potent as determined by IC₅₀measurements (FIG. 3, bottom). Similar results were seen for 2C2-YTE(data not shown).

2.2. Growth Suppression of MDA-MB-175 Breast Cancer Cells

MDA-MB-175 (ATCC No. HTB-25™) is an established HRG-expressing(γ-isoform) breast cancer cell-line that depends on HRG-HER3 signalingpathway for growth and survival. Cells were plated at a density of 2,000cells/well in a 96-well white-walled plate and were allowed to attachovernight. The following day, media was removed and replaced with 100μ/well fresh complete growth medium containing test and controlantibodies. Plates were then incubated for a total of 6 days. Tocalculate relative cell number, CellTiter-Glo™ (Promega, Madison, Wis.)was used according to manufacturer's protocol. After CellTiter-Glo™addition, plates were incubated at room temperature for 10 minutes andluminescence was measured using a microplate reader.

The growth assay was carried out with 2C2, MM, or AMG anti-HER3monoclonal antibodies. As shown in FIG. 4, all three antibodies achievedanti-proliferation effect to various extents, with 2C2 showing higherpotency (IC₅₀=0.14 μg/ml) (FIG. 4, top) and higher growth suppression(72%) (FIG. 4, bottom).

2.3. Growth Suppression of HMCB Melanoma Cells

HMCB (ATCC No. CRL-9607™) is an established HRG-expressing (1β-isoform)melanoma model driven by HRG-induced HER2-HER3 heterodimerization. HMCBcells were plated at a density of 750 per well in 100 μl of completemedium containing 10% heat-inactivated FBS in 96 well plates (Costar®).The next day, antibody treatments were prepared in complete medium. Thestarting concentration for all anti-HER3 monoclonal antibodies andcontrol IgG was 10 μg/ml, and serial dilutions were prepared in completemedium. The plating medium was removed and treatments were added in 100μl per well in triplicates.

Plates were then incubated in 5% CO₂ at 37° C. for 6 days. Equal volumesof CellTiter-Glo™ reagent were added to each well. Plates were rocked ona plate shaker for 10 minutes at room temperature to ensure completecell lysis. Luminescence was measured using a 2104 EnVision® MultilabelReader (PerkinElmer, Waltham, Mass.).

As shown in FIG. 5, 2C2 was again more potent than existing antibodies.2C2 was 8-30 fold more potent than the published anti-HER3 monoclonalantibodies AMG and MM in inhibiting cell growth of the HMCB melanomacell line.

2.4. HER3 and AKT Activity Assays in HMCB Melanoma Cells and A549 NSCLCCells

The ability of the HER3 leads to suppress the HER3 signaling pathway inthe HRG-autocrine HMCB (ATCC No. CRL-9607™) and A549 (ATCC No. CCL-185)models were assessed. HMCB cells were plated at 10⁵ per well in 24-wellplates and in medium containing 10% heat-inactivated FBS and allowed toreach a confluency of 80% or more prior to antibody treatment. Theplating medium was removed and the cells were subjected to incubationwith the antibodies. Anti-HER3 monoclonal antibodies and a control IgGwere prepared in complete medium. The starting concentration for allanti-HER3 antibodies was 10 μg/ml and serial dilutions were performed.The control IgG was only used at a concentration of 10 μg/ml. Treatmentswere applied following removal of plating medium. After an incubation of6 hours (HMCB cells) or 72 hours (A549 cells) in 5% CO₂ at 37° C., cellswere washed once with ice-cold PBS and then lysed by adding LaemmliReducing buffer (Boston BioProducts, Ashland, Mass.).

After a brief incubation, cell lysates were collected, equal amountswere loaded onto Bis NuPAGE® Novex® Bis-Tris gels (Invitrogen, Carlsbad,Calif.) and proteins transferred to PVDF membranes (Invitrogen,Carlsbad, Calif.). Membranes were blocked with 5% nonfat dry milk and0.1% Tween 20 (Sigma, St. Louis, Mo.) in TBS (pH 7.4) and incubatedovernight at 4° C. with antibodies to HER3 (sc-285 antibody, CellSignaling Technology, Beverly, Mass.), pHER3 (4791 antibody, CellSignaling Technology, Beverly, Mass.), AKT (9272 antibody, CellSignaling, Technology, Beverly, Mass.), pAKT (4060 antibody, CellSignaling Technology, Beverly, Mass.), neuregulin-1/HRG (NRG1/HRG)antibody (sc-348, Santa Cruz) and GAPDH (G8795 antibody, Sigma, St.Louis, Mo.).

Membranes were washed in 0.1% Tween 20 in TBS and then incubated for 1hour in horseradish peroxidase-conjugated streptavidin secondaryantibodies (GE Healthcare). After washing, protein bands were detectedin X-ray film by using SuperSignal® West Femto ChemiluminescentSubstrate and SuperSignal® West Pico Chemiluminescent Substrate(Pierce/Thermo Scientific, Rockford, Ill.).

As shown in FIGS. 6 and 7, the 2C2 antibody abrogated the HER3 signalingpathway in both HMCB and A549 cells. 2C2 efficiently suppressed pHER3and its downstream effector molecule pAKT in a dose-dependent manner andwas more potent than either of the published anti-HER3 monoclonalantibodies AMG or the MM in HMCB cells. The 2C2 antibody also surpressedpHER3 and its downstream effector molecule pAKT in A549 cells.

2.5. Assay for HER3 Phosphorylation (pHER3) in Cell Models for Lung,Gastric, and Breast Cancer

2.5.1. pHER3 Cell Assay

Cells (HCC827 NSCLC cells, Gefitinib-resistant HCC827 NSCLC cells, MKN45gastric cancer cells, Kato III gastric cancer cells, or BT-474HER2-amplified breast cancer cells) were plated at a density of 30,000cells/well in a 96-well plate and were allowed to attach overnight. Thecells were then treated with test or control antibodies at the indicateddose-curve at 37° C. for 4 hours. At the end of treatment, media wasremoved and cells were washed with PBS. Cells were lysed in 80 μl TritonX lysis buffer (Boston BioProducts, Ashland, Mass.) with protease andphosphatase inhibitors (Calbiochem, La Jolla, Calif.) and were stored at−20° C. until analysis. pHER3 ELISA was then performed followingmanufacturer's protocol (R&D Systems, DYC1769, Minneapolis, Minn.) usinghalf-volume 96-well ELISA plates (Costar 3690) and 50 μl of cell lysateper well.

2.5.2. Suppression of pHER3 Activity in HCC827 Cells

HCC827 cells (ATCC CRL-2868™), a mutant EGFR-driven non-small cell lungcancer (NSCLC) model, were treated with test or control antibodies asdescribed above in Example section 2.5.1 (see above). As shown in FIG.8A, the 2C2 antibody was able to partially inhibit pHER3 signal, whereasthe published anti-HER3 monoclonal antibodies AMG and MM were lesseffective and 10-fold less potent than 2C2.

2.5.3. Suppression of pHER3 Activity in Gefitinib-Resistant HCC827 Cells

HCC827 harbors and is driven by mutant-EGFR, which makes it highlysensitive to EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib.Parental HCC827 cells were exposed to a constant toxic dose of gefitiniband resistant clones were isolated that were shown to harbor amplifiedcMET, a known mechanism for cancers to escape TKI therapy. TKI-resistantHCC827 cells were treated with the anti-HER3 monoclonal antibodies asdescribed above in Examples section 2.5.1 (see above). As shown in FIG.8B, the anti-HER3 monoclonal antibody 2C2 suppressed HER3 activity inthe mutant HCC827 made resistant to gefitinib. Similar to the resultsseen for the parental cell line, 2C2 displayed higher potency than theAMG and MM antibodies (about 10-fold better potency) in theTKI-resistant HCC827 cell line.

2.5.4. Suppression of pHER3 Activity in MKN45 Cells

Even though cMET is not a member of the Her-family, it has been shown tobe capable of forming dimers with HER3. The MKN45 cMET-amplified gastriccancer model cell line was used to assess whether anti-HER3 antibodiescould antagonize cMET-driven HER3 activation. MKN45 cells were treatedwith the anti-HER3 monoclonal antibodies as described above in Examplessection 2.5.1. As shown in FIG. 8C, all three anti-HER3 monoclonalantibodies (2C2, AMG and MM) were able to suppress pHER3 in MKN45 cells,but 2C2 displayed higher potency than the AMG and MM antibodies(approximately 5-7-fold better potency).

2.5.5. Suppression of pHER3 Activity in Kato III Cells

Besides coupling with EGFR, HER2 and cMET, HER3 dimerizes with FGFR2 tofacilitate its transforming potential. The Kato III (ATCC No. HTB-103™)cell line, a FGFR2-amplifed gastric cancer model, was used to assesswhether anti-HER3 antibodies could suppress FGFR2-driven HER3activation. Kato III cells were treated with the anti-HER3 monoclonalantibodies as described above in Examples section 2.5.1 (see above). Inthis model, all three anti-HER3 monoclonal antibodies (2C2, AMG, and MM)achieved similar maximal extents of pHER3 suppression (˜60%), but asmeasured by IC₅₀, 2C2 was 15-20-fold more potent than the AMG and MMantibodies, respectively (FIG. 8D).

2.5.6. Suppression of pHER3 Activity in BT-474 Cells

HER2-HER3 dimers have been shown to be one of the most transformingoncogenic entities in cancer. Accordingly, we investigated the anti-HER3monoclonal antibodies in the BT-474 cell-line (ATCC NO. HTB-20™), awell-established HER2-amplified breast cancer model that does notexpress the ligand and is expected to be driven by ligand-independentHER2-HER3 dimerization. BT-474 cells were treated with the threeanti-HER3 monoclonal antibodies and also 2C2-YTE, as described above inExamples section 2.5.1. Unlike the models where all three anti-HER3monoclonal antibodies tested were active, such as HCC827 cells,Gefitinib-resistant HCC827 cells, MKN45 cells, and Kato III cells, 2C2(both parent 2C2 and 2C2-YTE mutant) was the only one among theanti-HER3 monoclonal antibodies tested showing substantial activitysuppressing pHER3 (FIG. 8E). These results indicated that 2C2 (bothparent 2C2 form and 2C2-YTE mutant) was functional in aligand-independent model and demonstrated the bi-functionality of 2C2 inboth ligand-dependent and ligand-independent settings.

2.6. Assay for AKT Phosphorylation (pAKT) in Cell Models for Gastric,and Breast Cancer

2.6.1. pAKT Cell Assay

Cells (MKN45 gastric cancer cells, Kato III gastric cancer cells, orBT-474 HER2-amplified breast cancer cells) were plated at a density of30,000 cells/well in 96-well plates and were allowed to attachovernight. The cells were then treated with test or control antibodiesat the indicated dose-curve at 37° C. for 4 hours. At the end oftreatment, media was removed and cells were washed with PBS. Cells werelysed in 80 μl of Triton X lysis buffer (Boston BioProducts, Ashland,Mass.) with protease and phosphatase inhibitors (Calbiochem, La Jolla,Calif.), and were stored at −20° C. until analysis. AKT/pAKT wereanalyzed based on the manufacturer's protocol included in the Phospho(Ser473)/Total AKT Whole Cell Lysate Kit (Cat. No. K15100D, Meso-ScaleDiscovery, Gaithersburg, Md.) to determine pAKT content.

2.6.2. Suppression of pAKT Activity in MKN45 Cells

To ascertain if 2C2 could suppress HER3 downstream signaling pathway inaddition to pHER3, we additionally assessed its ability to suppress AKTphosphorylation in the amplified cMET-driven gastric cancer model MKN45.MKN45 cells were treated with anti-HER3 monoclonal antibodies asdescribed above in Examples section 2.6.1. In this model system, the 2C2monoclonal antibody achieved partial pAKT inhibition with higher potency(approximately 5-7-fold higher) than the AMG, and MM anti-HER3monoclonal antibodies (FIG. 9A). This demonstrated that 2C2 not onlyinhibits HER3 activity but also suppresses downstream effector moleculesof HER3 such as pAKT.

2.6.3. Suppression of pAKT Activity in Kato III Cells

To investigate whether this activity translated into a better potencysuppressing pAKT, the effector of HER3, we analyzed pAKT inhibition byvarious anti-HER3 monoclonal antibodies using in this cell-line aMeso-Scale Discovery assay as described above in Examples section 2.6.1.As shown in FIG. 9B, consistent with the pHER3 data, 2C2 suppressed pAKTin amplified FGFR2-driven gastric cancer model Kato III cells. 2C2 againachieved higher potency (as measured by IC₅₀) and maximal response inpAKT inhibition than the AMG and MM antibodies.

2.6.4. Suppression of pAKT Activity in BT-474 Cells

HER2-HER3 dimers have been shown to be one of the most transformingoncogenic entities in cancer. Accordingly, we investigated the activityof anti-HER3 monoclonal antibodies in the BT-474 cell-line. BT-474 cellswere treated with the anti-HER3 monoclonal antibodies as described abovein Examples section 2.6.1, supra, and also with the YTE mutant form of2C2. Unlike the models where all three anti-HER3 monoclonal antibodiestested (2C2, AMG and MM) were active, such as MKN45 and KatoIII cells,2C2 (both parent 2C2 form and 2C2-YTE mutant) was the only one among theanti-HER3 monoclonal antibodies tested that showed substantial activitysuppressing pAKT (FIG. 9C). These results indicated that 2C2 (bothparent 2C2 form and 2C2-YTE mutant) was functional in aligand-independent model and demonstrated the bi-functionality of 2C2(both parent 2C2 form and 2C2-YTE mutant) in both ligand-dependent andligand-independent settings.

2.7. Suppression of HER3 Signaling and Cell Proliferation in MDA-MB-361Cells.

To characterize the activity of 2C2-YTE In HER2-amplified breast cancercells that are not highly responsive to trastuzumab, we focused onMDA-MB-361 (ATCC No. HTB-27), a breast cancer model that harbors theactivating mutation in PIK3CA (E545K), which may contribute to itsresistance to trastuzumab due to intrinsic activation of the PI3Kpathway (Junttila et al, 2009, Cancer Cell. 15:429-40). We determinedthe effects of 2C2 on HER3 signaling and cell proliferation in thismodel.

To assess signaling the human breast cell line MDA-MB-361 was plated in24-well plates at a density of 150,000 cells per well in RPMI(Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum(FBS) (Invitrogen). The next day, the plating medium was removed andcells were subjected to incubation with the anti-HER3 antibody 2C2 or acontrol antibody, in complete medium at a final concentration of 30μg/mL. After an incubation of 6 hours in 5% CO₂ at 37° C., cells werewashed once with ice-cold PBS and then lysed by adding Laemmli Reducingbuffer (Boston BioProducts, Ashland, Mass.). After a brief incubation,cell lysates were collected, equal amounts were loaded onto Bis NuPAGE®Novex® Bis-Tris gels (Invitrogen, Carlsbad, Calif.) and proteinstransferred to PVDF membranes (Invitrogen, Carlsbad, Calif.). Membraneswere blocked with 5% nonfat dry milk and 0.1% Tween 20 (Sigma, St.Louis, Mo.) in TBS (pH 7.4) and incubated overnight at 4° C. withantibodies to HER3 (sc-285 antibody, Cell Signaling Technology, Beverly,Mass.) and pHER3 (4791 antibody, Cell Signaling Technology, Beverly,Mass.). Membranes were washed in 0.1% Tween 20 in TBS and then incubatedfor 1 hour in horseradish peroxidase-conjugated streptavidin secondaryantibodies (GE Healthcare). After washing, protein bands were detectedin X-ray film by using SuperSignal® West Femto ChemiluminescentSubstrate and SuperSignal® West Pico Chemiluminescent Substrate(Pierce/Thermo Scientific, Rockford, Ill.).

To access cell proliferation MDA-MB-361 cells were seeded at a densityof 2,000 cells in 100 μL of medium containing 10% heat-inactivated FBS;Costar white polystyrene tissue-culture treated 96-well plates with flatbottoms (Corning) were used. The next day, the plating medium wasremoved and antibodies were added in complete medium to a final volumeof 100 μL per well. Plates were then incubated in 5% CO2 at 37° C. for 6or 14 days. For the 14-day assay, fresh antibodies were applied at Day7. Equal volumes of CellTiter-GloR reagent (Promega) were added to eachwell at the end of each time point. Plates were rocked on a plate shakerfor 10 minutes at room temperature to ensure complete cell lysis.Luminescence was measured using an EnVision 2104 Multilabel Reader(PerkinElmer).

2C2 reduced pHER3 levels (FIG. 10A) and suppressed cell growth (FIG.10B) of this cell line, suggesting 2C2-YTE not only is active intrastuzumab-sensitive cancers with HER2-amplification, but also activein HER2-amplified cancers that are less sensitive to trastuzumab due tomutations on PIK3CA.

2.8. Identification of Novel HRG-dependent Cancer Types

To identify additional novel HRG-dependent cancer times multiple lungsquamous cell carcinoma (SCC) cell lines were screened for HER3signaling activity and HRG expression. HARA-B (JCRB No. JCRB1080.1) andKNS-62 (JCRB No. IF050358) cell lines expressed significant levels ofHER3, HRG as well as pHER3 (data not shown). Accordingly, weinvestigated the activity of 2C2 in the HARA-B and KNS-62 cell-lines.The cells were treated with the anti-HER3 monoclonal antibodiesessentially as described above in Examples section 2.4 supra, 2C2 wasable to reduce pHER3 levels in the HARA-B cell line (FIG. 11) and theKNS-62 cell line (data not shown). As shown below (Examples, section5.4), 2C2-YTE demonstrated dose-dependent anti-tumor efficacy in thehuman squamous HARA-B NSCLC xenograft model. Thus, these criteria (i.e.,expression of HER3, HRG as well as pHER3) may be useful screening toolsto identify additional cancer types responsive to anti-HER3 antibodies,including for example 2C2, AMG, MM as described herein and others knownin the art (see for example International Patent PublicationsWO2011/136911, WO2012/019024, WO2010/022814).

2.9. HER2 is a Major Driver in Certain HRG-dependent Cancer Types

In the presence of the HER3 ligand heregulin (HRG), HER3 heterodimerizeswith EGFR or HER2, which leads to phosphorylation of HER3 andtransmission of an oncogenic signal via phosphoinositide 3 kinase (PI3K)and protein kinase B (PKB), also known as AKT. A collection of CRCmodels were characterized to determine which receptor tyrosine kinase,EGFR or HER2, is the major driver of signaling through HER3.Specifically, six different CRC tumor cell lines, SW620 (ATCCNo.CCL-227), SW480 (ATCC No. CCL-228), Colo205 (ATCC No.CCL-222), LOVO(ATCC No.CCL-229), HCT15 (ATCC No.CCL-225), and Caco-2 (ATCC No.HTB-37),were treated with antagonists of HER2 or EGFR alone or in combinationwith the HER3 antagonist 2C2. Briefly, cells were seeded into 24-wellplates at a density of 1.5×10⁵ cells per well. The next day, 2 identicalsets of cells were treated with the 10 μg/mL of the followingantibodies: 2C2 anti-HER3 antibody, the R347 control IgG antibody, theanti-HER2 antibody 2C4 (e.g., Patent Publication WO2001/00245), theanti-EGFR antibody cetuximab or the EGFR tyrosine kinase inhibitorgefitinib at 5 μM. After 5-6 hours of incubation at 37° C., HRG wasadded at 50 ng/mL into one set of cells for 15 minutes at 37° C. Allcells were then washed with cold PBS and lysed by the addition of 60 μLof 2× SDS (sodium dodecyl sulfate) sample buffer (Invitrogen). Lysateswere transferred to 1.5 mL tubes and boiled for 5 minutes followed bychilling on ice for 2 minutes. Equal volumes (20 μL) of protein sampleswere resolved in NuPAGE Novex Bis-Tris gels (Invitrogen) before transferto polyvinylidene fluoride (PVDF) membranes (Invitrogen). Membranes werewashed in Tris-buffered saline (KPL) containing 0.1% Tween 20 (Sigma)and incubated overnight at 4° C. with antibodies to HER3 (Santa CruzBiotechnology), pHER3-Tyr1289 (Cell Signaling Technology),phosphorylated AKT (protein kinase B (pAKT)) (Cell SignalingTechnology), phosphorylated ERK (mitogen-activated proteinkinase/extracellular signal-regulated kinase (pERK)) (Cell SignalingTechnology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)(Sigma). Membranes were washed in Tris-buffered saline (KPL) containing0.1% Tween 20 (Sigma) and then incubated for 1 hour in horseradishperoxidase-conjugated secondary antibodies (GE HealthCare). Afterwashing, protein bands were detected on X-ray film by using SuperSignalWest Pico Chemiluminescent Substrate (Pierce/Thermo Scientific).

As seen in FIG. 12, both the anti-HER3 and the anti-HER2 antibodiesreduced the levels of HER3, pHER3 and pAKT in ligand stimulated cellswhile EGFR antagonist such as cetuximab and gefitinib treatment had noeffect on these signaling molecules. These data demonstrate that HER2 isthe major driver of HRG-induced HER3 signaling in all the cancer modelstested.

Example 3 Mechanism of Action Studies for Anti-HER3 MonoclonalAntibodies

3.1. Clone 16 Partially Blocked Ligand-Binding to HER3

The efficacy of anti-HER3 monoclonal antibodies to block ligand-inducedHER3 activity can be due to their ability to directly block offligand-binding. To investigate this scenario, we established an in vitroHRG-HER3 binding assay by coating a plate with heregulin (HRG) andbinding labeled recombinant HER3 protein to it.

3.1.1. HRG-HER3 Binding Assay

Microplate wells were coated with 10 ng/ml heregulin (HRGβ1, Cat. No.377-HB, R&D Systems, Minneapolis, Minn.) overnight at 4° C. The nextday, plates were washed 4 times with PBST (PBS+0.05% Tween 20) andblocked in PBS+1 μg/ml BSA at room temperature for 1 hour. Duringblocking, serial dilutions of test antibodies (Clone 16, AMG, MM and apositive control anti-HER3 ligand blocking monoclonal antibody) wereprepared in a separate plate in PBSTB (PBS+0.05% Tween 20+0.1% BSA) andcombined with 5 μg/ml of recombinant HER3 (Cat. No. 348-RB, R&D Systems,Minneapolis, Minn.) at room temperature for 30 minutes. ELISA plateswere then washed 4× with PBST before addition of antibody-HER3 mixture.Plates were incubated at room temperature for 1 hour and weresubsequently washed 4 times with PBST. Anti-His HRP (Cat. No. 34460,Qiagen, Valencia, Calif.) was added at room temperature for 1 hour.Plates were washed 4 times with PBST followed by detection with TMB.Plates were read at 450 nm using a microplate reader. Representativeresults are shown in FIG. 13.

3.1.2. Results

The ability of several monoclonal antibodies (Clone 16, AMG, MM and apositive control anti-HER3 ligand blocking monoclonal antibody) tointerfere with the binding of HRG to HER3 was tested. The positivecontrol HER3 ligand-blocking monoclonal antibody, efficiently andcompletely suppressed the HER3 binding to HRG. In contrast, Clone 16(the parental lead for 2C2, see “Affinity Optimization” Examples section1.6 above) was only partially effective in disrupting this binding(approximately 30% maximum inhibition). The AMG and MM monoclonalantibodies showed similar weak, partial blocking effect (FIG. 13). Thesefindings showed that Clone 16 was unlikely to function as a directligand-blocking monoclonal antibody.

3.2. 2C2 Disrupts HER2-HER3 Dimerization

Due to its kinase-deficient nature, HER3 monomer is not active and itneeds to form heterodimers with other RTKs to be active. The HER2:HER3dimer has been shown to be the most oncogenic signaling species in bothligand-dependent and independent settings (Junttila et al, 2009, CancerCell. 15:429-40). The disruption of HER2-HER3 dimerization by 2C2 wasassessed using an HRG-induced HER2-HER3 dimer formation assay in T-47Dcells, a ligand-dependent breast cancer model showing HRG-inducedHER3-HER2 dimer formation, and in ligand-independent BT-474 cells. Theassay was based on HER3-HER2 co-immunoprecipitation.

3.2.1. Ligand-induced HER2-HER3 Dimerization Assay

T-47D cells (ATCC Cat. No. HTB-133™) were seeded at 1×10⁶/well in 6 wellplates overnight. Next morning, cells were treated with 2C2, CL16, AMGand MM monoclonal antibodies at a concentration of 5 μg/ml in full serumfor 2 hours at 37° C. Controls included no antibody treatment, ortreatment with control R347 IgG1. Treatment was followed by 50 ng/ml HRGtreatment for 10 minutes at 37° C. (including a control not treated withHRG). Cells were washed 3 times with cold PBS before adding 500 μl ofcell lysis buffer, including protease and phosphatase inhibitors (Sigma,St. Louis, Mo.). Cells were lysed on ice for at least 30 minutes.Lysates were harvested with a cell scraper. 50 μl of a protein A beadssolution containing 25 μl protein A beads conjugated with 1 μl ofanti-HER3 mAb (Cat. No. MAB3481, R&D Systems, Minneapolis, Minn.) wereadded to 500 μl of cell lysate and transferred to immunoprecipitation(IP) columns. The IP columns were rotated overnight at 4° C.Subsequently, the IP columns were spun down to remove the lysates, andthe beads were washed with cold cell lysis buffer. 50 μl of 2× SDSsample buffer containing 50 mM DTT were added to each IP column, andcolumns were boiled for 4 minutes. The bottom tip of each column wasremoved, and columns were spun down to collect the eluates. 20 μl ofeluate were separated using SDS-PAGE. Western blotting was performed forboth HER2 and HER3 (with anti-HER2 antibody Cat. No. OP15L, CalBiochem,La Jolla, Calif.; and anti-HER3 antibody Cat. No. SC-285, Santa CruzBiotechnology, Inc., Santa Cruz, Calif.).

3.2.2. Ligand-Independent HER2-HER3 Dimerization Assay

BT-474 cells (ATCC Cat. No. HTB-20™) were plated at a density of 1×106cells per well in a 6-well plate in complete RPMI 1640 cell culturemedia with 10% heat-inactivated FBS. The next day, plating medium wasremoved and replaced with fresh complete RPMI 1640 containing saturatingdose of testing antibodies. In this experiment, CL16, the precursorlower affinity version of 2C2, 2C2, and R347, a control IgG, were testedat a concentration of 5 μg/mL. Cells were incubated with antibodies for2 hours at 37° C. Then the medium was removed, and cells were washedonce with cold PBS. The crosslinker 3,3′-dithiobis[sulfosuccinimidylpropionate] (DTSSP) was added at a concentration of 2mM in 1 mL cold PBS. Cells were incubated for at least 1 hour on ice.Cells were then washed 3 times with cold PBS. Cell lysis buffer (500 μL)containing protease and phosphatase inhibitors was added and the cellswere placed on ice for at least 30 minutes to allow for lysis beforeharvesting with a cell scraper. HER2 and HER3 were immunoprecipitatedfrom cell lysates. Cell lysates (500 μL) were combined with 50 μLprotein A sepharose beads (50% slurry; Invitrogen) pre-conjugated to 1μg of HER3 MAb (MAB3481, R&D Systems) in a SigmaPrep spin column(Sigma). The mixture was incubated with rotation at 4° C. overnight. Thenext day, beads were separated from the cell lysate by centrifugation.Beads in the columns were washed four times with cold cell lysis buffer(Cell Signaling Technologies) containing protease (Sigma) andphosphatase inhibitors (EMD Millipore). After the wash procedure, 50 μL2× SDS (sodium dodecyl sulfate) sample buffer containing 50 mMdithiothreitol (DTT; EMD Chemicals) was added into the spin columns. Thecolumns were then boiled for 4 minutes. Proteins were eluted bycentrifugation and used immediately for immunoblotting (as done insection 2.4).

3.2.3. Results

T-47D cells treated or not treated with HRG were lysed. HER3 wasprecipitated with anti-HER3 monoclonal antibody, then the proteins inthe pellet were resolved on SDS-PAGE and blotted for the presence ofHER2 as signs of HER2-HER3 interaction. The model was ligand-induciblesince the dimer only occurred after ligand-stimulation. A pre-treatmentwith 2C2 efficiently prevented dimer formation, demonstrating itsability to impede ligand-induced HER2-HER3 dimer formation. Otheranti-HER3 monoclonal antibodies including MM, AMG, and the parentalClone 16, were also found to be effective (FIG. 14A). When thecross-linker DTSSP was used to biochemically stabilize proteincomplexes, constitutive HER2:HER3 heterodimer was captured in theabsence of HRG in BT-474 cells, indicating a ligand-independentheterodimer formation. Pretreatment of cells with 2C2 or CL16effectively disrupted this heterodimer formation (FIG. 14B).

3.3. HER3 Internalization and Degradation Induced by 2C2

Target internalization and degradation are two common mechanisms bywhich monoclonal antibodies inhibit their target functions. First, weassessed the 2C2-mediated HER3 internalization in the BT-474 breastcancer cells. Next, we ascertained if this rapid 2C2-induced HER3internalization could be followed by target degradation.

3.3.1. HER3 Internalization Assay

HER3 internalization was determined using a Fluorescence Activated CellSorting (FACS) assay. BT-474 cells were detached with Accutase enzymeand suspend the cells in PBS containing 1% BSA (FACS buffer) to a celldensity of 10×10⁶ cells/ml. 50 μl of cells were added to each cell of aU-bottom 96 well plate. 50 μl of anti-HER3 monoclonal antibodies plusIsotope control (at 20 μg/ml) were added into each well to achieve a 10μg/ml final concentration. The plate was incubated at 37° C. for 0.5hours, 2 hours and 4.5 hours, respectively. Cells were washed with coldFACS buffer twice (cells were pelleted by centrifugation at 1,500 rpmfor 2 minutes). Cells were resuspended with cold FACS buffer containingmouse anti-human HER3 monoclonal antibody (Cat. No. MAB3481, R&DSystems, Minneapolis, Minn.) at 1 μg/ml or 10 μg/ml.

Cells and anti-human HER3 were incubated on ice for 1 hour. Cells werethen washed twice with cold FACS buffer. Cells were subsequentlyresuspended with cold FACS buffer containing an Alexa Fluor® 488-labeledsecondary antibody (Invitrogen) (1:200 v/v), and incubated on ice for30-45 minutes. Cells were then washed with cold FACS buffer twice andresuspended with 100 μl of cold FACS buffer. At this point, FACS wasperformed. Absolute Geometric Mean of Fluorescence Intensities (GMFI)were obtained by subtracting the GMFI from controls including only thesecondary antibody. Relative HER3 surface clearance was calculated bycomparing with results obtained using an IgG control monoclonalantibody. Representative results are shown in FIG. 15A.

3.3.2. HER3 Protein Degradation Assay

Lovo, HCT15 and SW620 colorectal model cancer cells (ATCC Nos. CCL-229,CCL-225 and CCL-227, respectively) were seeded at 1.5×10⁵/well in 24well plates. After overnight attachment, the cells were treated with 2C2and control monoclonal antibody for 3-4 hours. Cells were washed withcold PBS once, directly lysed with 50-60 μl of 2× SDS sample buffer andboiled at 100° C. for 10 minutes. 20 μl of samples were loaded intoSDS-PAGE gels, electrophoretically separated, and Western blotted withantibody against HER3 (Santa Cruz Biotech) to quantitate total HER3protein levels. Antibodies against GAPDH (Sigma) were also used toquantitate GAPDH levels as a general protein loading control.

3.3.3. Results

As shown in FIG. 15A, both doses of 2C2 had a very similar impact. A30-minute treatment caused a 39% loss of surface HER3 population (61%remaining), whereas a 2-hour treatment caused a 62% loss (38%remaining), suggesting a rapid target internalization by 2C2.Additionally, when the three different colorectal cancer models wereincubated with 2C2, complete HER3 degradation was observed in SW620cells, whereas nearly complete degradation was observed in the other twocell-lines (FIG. 15B), demonstrating that 2C2 was capable of strongtarget degradation capacity.

3.4. Effector Functions: Antibody-Dependent Cell-Mediated Cytotoxicity(ADCC) and Complement-Dependent Cytotoxicity (CDC)

ADCC is one recognized way through which a monoclonal antibody canconfer its anti-tumor efficacy in vivo. To assess the ADCC activity ofClone 16, we used an in vitro PBMC-enabled ADCC assay in twoHER2-amplfied breast cancer models: BT-474 and SkBR3.Herceptin/Trastuzumab was used as positive control since it has beenshown to confer ADCC effect in these type of cancers. In both models weobserved significant tumor-killing effects from Herceptin, but theremaining monoclonal antibodies tested, Clone 16, AMG and MM, werelargely inactive, indicating that they lacked appreciable ADCC effect(data not shown). 2C2-YTE was tested in CDC assays using human serum asa source of complement. In addition, the anti-HER2 antibody trastuzumaband the anti-CD20 antibody rituximab, were included as controls. None ofthe antibodies including 2C2-YTE showed any detectable CDC activity atany concentration (data not shown). SkBR3 cells do not express CD20. Asa positive control, rituximab demonstrated substantial cell-killactivity in a similar CDC assay against Daudi cells, which express CD20(data not shown).

3.5. Cell-Cycle Arrest

3.5.1. Cell-cycle Arrest Assay in SkBR3 Breast Cancer Cells

BioSantecells (ATCC No. HTB-30) were plated at a density of 150,000cells/well in a 6-well plate and allowed to attach overnight. Thefollowing day, media was removed and replaced with fresh growth mediumcontaining test and control antibodies. Cells were then incubated at 37°C. for 48 hours. At the end of the treatment, cells were trypsinized,pooled into a 15 ml conical tube, and centrifuged at 1500 rpm for 5minutes. Cell were then washed once with PBS and fixed in ice cold 70%ethanol at −20° C. overnight.

Following fixation, cells were centrifuged as described above, washedonce in PBS, and resuspended in staining solution (PBS+0.1% TritonX-100, 0.2 mg/ml DNAse-free RNAse A, and 20 μg/ml propidium iodide).Cells were stained for 30 minutes at room temperature in the dark, andanalyzed using an LSRII Flow Cytometer System (BD Biosciences).Propidium iodide was detected using the Texas Red channel; data wasanalyzed using the FlowJo flow cytometry analysis package (Tree Star,Inc., Oreg.) using the Dean/Jett/Fox Model.

3.5.2. Results

The FACS-based cell-cycle analysis showed that in SkBR3 cells, aHER2-amplified breast cancer cell-line similar to BT-474, both Herceptinand Clone 16 (parental lead for 2C2) caused cell-cycle arrest atG1-phase (increased G1-population by decreasing S/G2 populations asshown in FIG. 16).

3.6. Anti-angiogenic Effects by Blocking HRG-induced VEGF Secretion

HRG has been shown to drive secretion of VEGF, a major pro-angiogeniccytokine, in various cancer models. Therefore we assessed the inhibitoryeffects of 2C2 in suppressing HRG-induced VEGF secretion in two breastcancer models: MCF-7 and BT-474.

3.6.1. HRG-induced VEGF Secretion Assay

MCF-7 cells and BT-474 cells were plated at a density of 100,000cells/well in a 24-well plate, and were allowed to rest for 2 days.Media was then removed and replaced with 500 μl of fresh growth mediumcontaining 2% FBS and control and test antibodies. Following 24 hourincubation, cell culture media was collected and VEGF levels weredetermined using a VEGF ELISA Kit (R&D Systems DY293B). Relative cellnumber in each well was determined by adding fresh media to the cellsalong with Cell Titer Glo (Promega, 1:1 ratio) and incubating plates for10 minutes at room temperature. Luminescence was read using a platereader, and these values were used for normalization of the data.

3.6.2. Results

HRG treatment induced dramatic increases in VEGF secretion in the BT-474(FIG. 17A) and MCF-7 (FIG. 17B) both breast cancer model cell-linesranging from 6.5-fold to 8-fold. CL16 (Clone 16), and MM monoclonalantibodies were able to suppress most of the increases, suggesting thatthese anti-HER3 monoclonal antibodies can confer additional vascularmodulation effects.

Example 4 Cross Reactivity with Cynomolgus Monkey and Mouse HER3

4.1. 2C2 Binds to Cynomolgus and mouse HER3 with Similar Affinity as toHuman HER3

Biacore assays were performed essentially as described above to comparethe affinity of 2C2 to human, cynomolgus monkey (cyno) and mouse HER3 toenable relevant toxicity species selection (top portion of Table 7.)Additional Biacore assays were performed for 2C2-YTE using a higherresolution BIAcore instrument, an alternative Fc capture reagent and arefined injection protocol to correct for background binding. Briefly,Protein A capture reagent was immobilized onto two adjacent flow cellsconnected in series on the same CMS sensor chip, using a standard amineprotocol as outlined by the instrument's manufacturer. One of theseProtein A surfaces was used as a reference surface for this experiment,while the other served as the active surface used to record IgG captureand subsequent HER3 (ECD) binding. The final Protein A densities on thereference and active flow cell surfaces were recorded as 1986 RUs and1979 RUs, respectively. As configured, the method was set up such that2C2-YTE IgG was first captured onto only the active Protein A surface,followed by an injection of a HER3 protein solution over both the activeand reference flow cell surfaces. In so doing, this strategy correctsthe binding curve for any non-specific binding of the HER3 analyte tothe Protein A capture surface. For the IgG capture step, 2C2-YTE IgG wasprepared at 10 nM in HBS-EP+instrument buffer (0.01 M HEPES, pH 7.4,0.15 M NaCl, 3 mM EDTA, and 0.05% P20), then injected over the activeProtein A flow cell surface for 30 seconds at a flow rate of 10 μL/min.Human, cyno, and murine HER3 protein were then initially prepared at 500nM stock solutions in instrument buffer, then two-fold serial dilutionseries of each were generated to provide a final concentration of 0.39nM. The HER3 protein was then injected over both the active andreference cell Protein A surfaces for 120 seconds, at a flow rate of 75μL/min. Dissociation data was collected for 15 minutes, followed by two60-second pulses with 10 mM Gly buffer, pH 1.7, between injections toregenerate the flow cells back to the Protein A capture surfaces.Several buffer injections were also interspersed throughout theinjection series. Select buffer injections were subsequently used alongwith the reference cell data to correct the raw data sets for injectionartifacts and/or non-specific binding interactions, a technique commonlyreferred to as “double-referencing” (Myszka, 1999). Fully correctedbinding data were then globally fit to a 1:1 binding model(BIAevaluation 4.1 software) that included a term to correct for masstransport-limited binding, should it be detected. This analysisdetermined the kinetic rate (kon, koff) constants, from which theapparent KD was then calculated as koff/kon (bottom of Table 7). Thevariation in the K_(on) and K_(off) values between the two sets ofexperiments are likely due to the differences between the two protocolsas detailed above and were generally within the accepted two fold errorrange for measuring these kinetic parameters. As shown in TABLE 7, theaffinity of 2C2, and 2C2-YTE to cyno HER3 was virtually identical withthat to human HER3. The affinity for mouse HER3 was within 3-fold of theaffinity for human HER3.

TABLE 7 Biacore binding assay showing 2C2's affinity to human, cyno, andmouse HER3. 2C2 2C2 2C2 IgG Capture (exp34c, 34d, 43b) (exp43d) (exp43f)Receptor huHER3 (ECD)-His muHER3-His Cyno HER3-His Format IgG (Fc)capture IgG (Fc) capture IgG (Fc) capture K_(on) (1/Ms) 4.27 (+/−0.45)3.26 4.66  (×10⁵) K_(off) (1/s) 1.71 (+/−0.18) 3.78 1.81  (×10⁻⁴) K_(D)(nM) 0.402 (+/−0.029) 1.16 0.389 IgG Capture 2C2-YTE 2C2-YTE 2C2-YTEReceptor huHER3 (ECD)-His muHER3-His Cyno HER3-His Format IgG (Fc)capture IgG (Fc) capture IgG (Fc) capture K_(on) (1/Ms) 1.61  1.11 1.52 (×10⁵) K_(off) (1/s) 0.743 1.91 0.734 (×10⁻⁴) K_(D) (nM) 0.461  1.7210.483

4.2. Assay for HRG-induced Phosphorylation of Cynomolgus HER3

Ad293 cells (Stratagene No. 240085) were transiently transfected withfull length cynoHER3-expression vector following protocol provided withthe Lipofectamine 2000 reagent (Invitrogen). Cells were allowed toincubate at 37° C. for 48 hours before treatment. Antibodies were addedat 10 μg/ml in complete growth medium for 1 hour followed by stimulationwith 20 ng/ml HRGβ1 (R&D Systems) for 10 minutes at 37° C. At the end oftreatment, media was removed and cells were washed once with PBS. Cellswere lysed with 2× Novex Tris-glycine sample buffer (Invitrogen) and thelevels of pHER3 and total HER3 were determined by immunoblotting (CellSignaling antibody #4791 and Santa Cruz antibody #285, respectively).Densitometry of bands was accomplished using ImageJ software (NIH,imagej.nih.gov/ij/).

4.3. Results

To fully establish the binding and cross-modulation of cyno HER3 by 2C2,a stable Ad293 cell-line ectopically expressing full-length cyno HER3was established, as demonstrated by Western Blot (FIG. 18A). Whentreated with HRG, the cyno HER3 underwent robust activation as evidencedby the induction of pHER3 signal (FIG. 18B). When cells were co-treatedwith 2C2 but not when they were treated with the R347 control antibody,pHER3 induction was completely abrogated, demonstrating that 2C2 was notonly able to bind to cyno HER3 on cell-surface, but also able toefficiently ablate its activation (FIG. 18B). Combined with the aboveBiacore affinity measurement data showing that 2C2 displayed identicalaffinity to cyno HER3 as to human HER3, these results validated cyno asa relevant toxicity species for 2C2 trials.

In Vivo studies for Anti-HER3 Monoclonal Antibodies

4.4. Subcutaneous Human FADU Head and Neck Xenograft Model Studies

4.4.1. Method

Human FADU Head and Neck cells (ATCC No.HTB-43) were maintained at 37°C. in a 5% CO₂ incubator in RPMI 1640 medium containing 4.5 g/L glucose,L-glutamine, sodium pyruvate and 10% fetal bovine serum. Xenografts wereestablished by subcutaneously injecting 5×10⁶ cells per mouse (suspendedin 50% matrigel) into the right flanks of 4- to 6-week-old athymic nu/numice. Tumors were allowed to grow up to 200 mm³ before randomization forefficacy studies. 2C2, 2C2-YTE, cetuximab, control IgG1 or thecombination of 2C2 with cetuximab monoclonal antibodies wereadministered intraperitoneally. For dose dependency studies the 2C2 wasadminstered at 3, 5, 7, and 10 mg per kilogram body weight (mg/kg), thecontrol at 10 mg/kg. For the combination studies 2C2 was administered at3 mg/kg, cetuximab at 30 mg/kg and the control antibody at 6 mg/kg.Caliper measurements were used to calculate tumor volumes using theformula:

tumor volume=π÷6(length×width×width)

-   -   for tumors grown in mice. Antitumor effects are expressed as        percent delta tumor growth inhibition (TGI), which was        calculated as follows:

percent delta TGI=1−(dT÷dC)×100,

-   -   where dT=change in mean tumor volume in treatment group compared        to the value at staging, and dC=change in mean tumor volume in        control group compared to the value at staging.

At the conclusion of the efficacy studies with 2C2, mice were treatedwith 2C2 a final time as indicated to determine pharmacokinetic values.Cardiac puncture was performed to collect blood into Microtainer SerumSeparator Tubes (SST). Tubes with blood were vortexed gently for 10seconds and kept at room temperature for 20 minutes to allow the serumto clot. Samples were centrifuged at 1000×g for 10 minutes, and theserum samples were carefully transferred into new tubes and stored at−80° C.

An indirect Enzyme-Linked Immunosorbent Assay (ELISA) format was usedfor the quantitative determination of 2C2 in mouse serum. Standards,quality controls, and mouse serum samples were incubated with goatanti-human IgG antibodies which were immobilized on a 96-well microtiterplate. After incubation, unbound materials were removed by a wash stepand 2C2 was detected using a goat anti-human IgG withhorseradish-peroxidase conjugate. An acidic stopping solution was addedand the degree of enzymatic turnover of substrate was determined bymeasuring absorbance at 450 nm. The absorbance measured was directlyproportional to the concentration of 2C2 present in the mouse serum. A2C2 standard curve for the assay was used to interpolate theconcentration of the serum samples.

4.4.2. Results.

Utilizing a human FADU Head and Neck xenograft model grownsubcutaneously in female nude mice, 2C2 demonstrated dose-dependentanti-tumor efficacy. Maximal efficacy at 99% tumor growth inhibition(dTGI) was observed with 7 mg/kg administered twice per week for theduration of the study (FIG. 19A).

Combined administration of 3 mg/kg of 2C2 with 30 mg/kg of cetuximabadministered two times per week during the treatment phase (days 7-18)showed clear synergistic anti-tumor efficacy in the FADU xenograft model(FIG. 19B). This effect was long lasting and the tumors only started togrow back at the end of the regrowth phase at day 40. The combinationtreatment produced 7 out of 10 partial regressions and 2/10 completeregressions.

2C2 cross-reacted with mouse HER3 and it is well established that HER3is expressed in many non-diseased mouse tissues. Therefore, host HER3could serve as a sink to absorb the 2C2 monoclonal antibody before itgets to the tumor tissue. Using tumor-bearing female nude mice, 2C2 at 5mg/kg was administered either once or three times to these mice and theexposure levels of 2C2 were followed over time. 2C2 was only detectable1 day after the last dose of 5 mg/kg of 2C2 and became undetectableafter 3 days after the last treatment (FIG. 36). On the other hand,dosing with 30 mg/kg of 2C2 using the same schedules as for 5 mg/kg ledto a much more prolonged window where 2C2 could be measured in mouseserum. These findings demonstrated non-linear pharmacokinetics for 2C2after single dose and repeat-dose administration of 5 mg/kg or 30 mg/kgto tumor-bearing mice. The data showed that mouse HER3 can act as a sinkto bind 2C2 administered to the mice and that 30 mg/kg as a single dosewas sufficient to saturate the sink.

The existence of a HER3 sink in mice for 2C2 was confirmed functionallyby administering a high loading dose of 2C2 follow by a low maintenancedose in mice with FADU xenograft tumors. The anti-tumor efficacy of a 10mg/kg loading dose and a 3 mg/kg maintenance dose of 2C2 wasdemonstrated in the FADU tumor model. 10 mg/kg of 2C2 as a single dosehad only transient anti-tumor efficacy. 2C2 given at 3 mg/kg twice perweek had modest but continuous efficacy. The combination of the 10 mg/kgloading dose with the 3 mg/kg maintenance dose of 2C2 was moreefficacious in blocking tumor growth compared to either treatmentschedule alone (FIG. 21).

The ability of 2C2 to modulate the pharmacodynamic markers pHER3 andpAKT was tested in FADU xenograft tumor extracts. 2C2 was administeredtwice at 30 mg/kg within 48 hours to mice bearing human FADU xenografttumors and extracts were analyzed 24 hours later. Briefly, athymic nudemice were implanted subcutaneously with FADU head and neck cancer cells.Animals were administered 2C2 at 30 mg/kg twice within 48 hours.Extracts were prepared 24 hours later for analysis of pHER3, pAKT andtotal HER3 (FIG. 22, top, middle and bottom panels, respectively). R347was used as the control IgG1 antibody. There were 6 animals pertreatment group. Data are presented as the mean±standard deviation.Here, 2C2 inhibited phosphorylation of both HER3 and AKT by 59.5% and51.7%, respectively, compared to tumors from control IgG1-treated mice(FIG. 22, top and middle panels). No modulation of total HER3 wasobserved by 2C2 (FIG. 22, bottom panel).

4.5. Subcutaneous Human Detroit562 Head and Neck Xenograft Model Studies

4.5.1. Method.

Human Detroit562 Head and Neck cells (ATCC No. CCL-138) were maintainedat 37° C. in a 5% CO₂ incubator in RPMI 1640 medium containing 4.5 g/Lglucose, L-glutamine, sodium pyruvate and 10% fetal bovine serum.Xenografts were established by subcutaneously injecting 5×10⁶ cells permouse into the right flanks of 4- to 6-week-old athymic nu/nu mice.Tumors were allowed to grow up to 200 mm³ before randomization forefficacy studies. 2C2, 2C2-YTE, cetuximab, control IgG1 or thecombination of 2C2 with cetuximab monoclonal antibodies wereadministered intraperitoneally. For dose dependency studies the 2C2 wasadministered at, 1, 3,10, and 30 mg per kilogram body weight (mg/kg).For the combination studies 2C2 was administered at 3 mg/kg, cetuximabat 30 mg/kg and the control antibody at 10 mg/kg. Caliper measurementswere used to calculate tumor volumes using the formula:

tumor volume=π÷6(length×width×width)

-   -   for tumors grown in mice. Antitumor effects are expressed as        percent delta tumor growth inhibition (TGI), which was        calculated as follows:

percent delta TGI=1−(dT÷dC)×100,

-   -   where dT=change in mean tumor volume in treatment group compared        to the value at staging, and dC=change in mean tumor volume in        control group compared to the value at staging.

At the conclusion of the efficacy studies with 2C2, mice were treatedwith 2C2 a final time as indicated to determine pharmacokinetic values.Cardiac puncture was performed to collect blood into SST microtainertubes. Tubes with blood were vortexed gently for 10 seconds and kept atroom temperature for 20 minutes to allow the serum to clot. Samples werecentrifuged at 1000×g for 10 minutes, and the serum samples werecarefully transferred into new tubes and stored at −80° C.

An indirect Enzyme-Linked Immunosorbent Assay (ELISA) format was usedfor the quantitative determination of 2C2 in mouse serum. Standards,quality controls, and mouse serum samples were incubated with goatanti-human IgG antibodies which were immobilized on a 96-well microtiterplate. After incubation, unbound materials were removed by a wash stepand 2C2 was detected using a goat anti-human IgG withhorseradish-peroxidase conjugate. An acidic stopping solution was addedand the degree of enzymatic turnover of substrate was determined bymeasuring absorbance at 450 nm. The absorbance measured was directlyproportional to the concentration of 2C2 present in the mouse serum. A2C2 standard curve for the assay was used to interpolate theconcentration of the serum samples.

4.5.2. Results

2C2 showed anti-tumor efficacy in the human Detroit562 Head and Neckxenograft model grown subcutaneously in female nude mice. 10 mg/kg of2C2 administered twice per week was maximally efficacious at 72% dTGI(FIG. 23A). The Detroit562 model contains a PIK3CA mutation.

The Detroit562 tumor model was sensitive to the anti-EGFR monoclonalantibody cetuximab which caused tumor growth inhibition at 10 mg/kgadministered twice per week. The combination of 3 mg/kg of 2C2 with 10mg/kg of cetuximab added to the anti-tumor efficacy of cetuximab andresulted in 9 out of 10 partial regressions while cetuximab aloneproduced 5/10 partial regressions (FIG. 23B).

4.6. Subcutaneous Human CAL27 Head and Neck Xenograft Model Studies

4.6.1. Method.

Human CAL27 Head and Neck cells (ATCC No. CRL-2095) were maintained at37° C. in a 5% CO₂ incubator in RPMI 1640 medium containing 4.5 g/Lglucose, L-glutamine, sodium pyruvate and 10% fetal bovine serum.Xenografts were established by subcutaneously injecting 5×10⁶ cells permouse into the right flanks of 4- to 6-week-old athymic nu/nu mice.Tumors were allowed to grow up to 200 mm³ before randomization forefficacy studies. 2C2-YTE, cetuximab or control IgG1 were administeredintraperitoneally. For dose dependency studies the 2C2-YTE wasadminstered at 3, 10, and 30 mg per kilogram body weight (mg/kg).Caliper measurements were used to calculate tumor volumes using theformula:

tumor volume=π÷6(length×width×width)

-   -   for tumors grown in mice. Antitumor effects are expressed as        percent delta tumor growth inhibition (TGI), which was        calculated as follows:

percent delta TGI=1−(dT÷dC)×100,

-   -   where dT=change in mean tumor volume in treatment group compared        to the value at staging, and dC=change in mean tumor volume in        control group compared to the value at staging.

4.6.2. Results.

Dose-dependent activity of 2C2-YTE was confirmed in a third head andneck tumor model, CAL27, using 2C2-YTE. 2C2-YTE at 3, 10 or 30 mg/kgadministered twice per week showed TGI with 26.4%, 55.2%, or 68.8%,respectively, compared to control IgG1-treated animals (FIG. 24).

The CAL27 tumor model was sensitive to the anti-EGFR monoclonal antibodycetuximab which caused tumor growth inhibition at 30 mg/kg administeredtwice per week with TGI of 75.0% (FIG. 24).

4.7. Subcutaneous Human KRAS Mutated A549 NSCLC Xenograft Model Studies

4.7.1. Method

Human A549 NSCLC cells (ATCC No.CCL-185) which contain a mutation incodon 12 of the KRAS gene (were maintained at 37° C. in a 5% CO₂incubator in HAM'S F12K medium containing 4.5 g/L glucose, L-glutamine,sodium pyruvate and 10% fetal bovine serum. Xenografts were establishedby subcutaneously injecting 5×10⁶ cells per mouse (suspended in 50%matrigel) into the right flanks of 4- to 6-week-old athymic nu/nu mice.Tumors were allowed to grow up to 200 mm³ before randomization forefficacy studies. 2C2, 2C2-YTE, cetuximab, control IgG1 or thecombination of 2C2 with cetuximab monoclonal antibodies wereadministered intraperitoneally. For dose dependency studies the 2C2 wasadminstered at, 3, 10 and 30 mg per kilogram body weight (mg/kg) and2C2-YTE at 10 mg/kg. For the combination studies 2C2 and cetuximab wereeach administered at 10 mg/kg. Caliper measurements were used tocalculate tumor volumes using the formula:

tumor volume=π÷6(length×width×width)

-   -   for tumors grown in mice. Antitumor effects are expressed as        percent delta tumor growth inhibition (TGI), which was        calculated as follows:

percent delta TGI=1−(dT÷dC)×100,

-   -   where dT=change in mean tumor volume in treatment group compared        to the value at staging, and dC=change in mean tumor volume in        control group compared to the value at staging.

4.7.2. Results.

2C2 demonstrated dose-dependent anti-tumor efficacy in the human A549NSCLC xenograft model grown subcutaneously in female nude mice. Maximalefficacy of 91% dTGI was achieved with 30 mg/kg of 2C2 administeredtwice per week until day 33 (FIG. 25A). 2C2 and 2C2-YTE given at 10mg/kg displayed similar anti-tumor efficacy in this A549 tumor model.Once the treatment was stopped the tumors started to grow at the samerate as tumors in control-treated mice. The A549 xenograft modelcontains a KRAS mutation and a LKB-1 deletion.

Cetuximab at 10 mg/kg alone was not efficacious in this A549 tumormodel. However, the addition of cetuximab at 10 mg/kg to 2C2 also at 10mg/kg resulted in additive anti-tumor efficacy during the treatmentphase compared to 2C2 alone. In addition, the combination treatmentgroup showed a slower regrowth rate of the tumors after cessation oftreatment (FIG. 25B).

4.8. Subcutaneous Human HARA-B Squamous NSCLC Xenograft Model Studies

4.8.1. Method

Human squamous HARA-B NSCLC cells which express the wild-type RAS gene,HRG and pHER3 were maintained at 37° C. in a 5% CO₂ incubator in RPMI1640 medium containing 4.5 g/L D. glucose, 2.383 g/L HEPES Buffer, L.Glutamine, 1.5 g/L Sodium Bicarbinate, 110 mg/L sodium pyruvate and 10%fetal bovine serum. Xenografts were established by subcutaneouslyinjecting 5×10⁶ cells per mouse (suspended in 50% matrigel) into theright flanks of 4- to 6-week-old athymic nu/nu mice. Tumors were allowedto grow up to 227 mm³ before randomization for efficacy studies. 2C2-YTEwere administered intraperitoneally at 3, 10 and 30 mg per kilogram bodyweight (mg/kg), the control was at 30 mg/kg. Caliper measurements wereused to calculate tumor volumes using the formula:

tumor volume=π÷6(length×width×width)

-   -   for tumors grown in mice. Antitumor effects are expressed as        percent delta tumor growth inhibition (TGI), which was        calculated as follows:

percent delta TGI=1−(dT÷dC)×100,

-   -   where dT=change in mean tumor volume in treatment group compared        to the value at staging, and dC=change in mean tumor volume in        control group compared to the value at staging.

4.8.2. Results.

2C2-YTE demonstrated dose-dependent anti-tumor efficacy in the humansquamous HARA-B NSCLC xenograft model grown subcutaneously in femalenude mice. Maximal efficacy of 64.6% dTGI was achieved with 30 mg/kg of2C2-YTE administered twice per week until day 29 (FIG. 26). 2C2-YTEgiven at 10 mg/kg displayed similar anti-tumor efficacy as 30 mg/kg;however, 2C2-YTE at 3 mg/kg was not efficacious in this HARA-B tumormodel. The HARA-B xenograft model contains a wild-type RAS allele.

4.9. Subcutaneous Human HT-29 CRC Xenograft Model Studies

4.9.1. Method

Human HT-29 colorectal carcinoma cells (ATCC No. HTB-38) were maintainedat 37° C. in a 5% CO₂ incubator in RPMI 1640 medium containing 4.5 g/Lglucose, L-glutamine, sodium pyruvate and 10% fetal bovine serum.Xenografts were established by subcutaneously injecting 5×10⁶ cells permouse into the right flanks of 4- to 6-week-old athymic nu/nu mice.Tumors were allowed to grow up to 200 mm³ before randomization forefficacy studies. 2C2, 2C2-YTE and control IgG1 monoclonal antibodieswere administered intraperitoneally. 2C2 was administered at 2, 10 and30 mg per kilogram body weight (mg/kg), while 2C2-YTE was at 30 mg/kg.Caliper measurements were used to calculate tumor volumes using theformula:

tumor volume=π÷6(length×width×width)

-   -   for tumors grown in mice. Antitumor effects are expressed as        percent delta tumor growth inhibition (TGI), which was        calculated as follows:

percent delta TGI=1−(dT÷dC)×100,

-   -   where dT=change in mean tumor volume in treatment group compared        to the value at staging, and dC=change in mean tumor volume in        control group compared to the value at staging.

4.9.2. Results.

2C2 showed dose-dependent anti-tumor efficacy using the human HT-29colorectal xenograft model subcutaneously injected into female nudemice. 30 mg/kg of 2C2 administered twice per week was maximallyefficacious at 56% dTGI during the treatment phase (FIG. 27). 2C2-YTEdisplayed the same efficacy as 2C2 both given at 30 mg/kg. Once thetreatment was stopped the tumors grew at the same rate as the controltumors. The HT-29 xenograft model contains a BRAF mutation. Cetuximab at10 mg/kg alone had no measurable anti-tumor activity in this model. Theactivity of 2C2 30 mg/kg in combination with cetuximab at 10 mg/kg wasindistinguishable from the activity of 2C2 30 mg/kg alone at the end oftreatment phase (data not shown). This indicates that thisEGFR-expressing CRC tumor model, which responds well to 2C2, was notfurther inhibited by the addition of 2C2-YTE to cetuximab.

4.10. Subcutaneous Human HCT-116 CRC Xenograft Model Studies

4.10.1. Method

Human HCT-116 colorectal carcinoma cells were maintained at 37° C. in a5% CO₂ incubator in RPMI 1640 medium containing 4.5 g/L glucose,L-glutamine, sodium pyruvate and 10% fetal bovine serum. Xenografts wereestablished by subcutaneously injecting 5×10⁶ cells per mouse into theright flanks of 4- to 6-week-old athymic nu/nu mice. Tumors were allowedto grow up to 200 mm³ before randomization for efficacy studies. 2C2,2C2-YTE, cetuximab and control IgG1 monoclonal antibodies wereadministered intraperitoneally. 2C2 was administered at 3, 10 and 30 mgper kilogram body weight (mg/kg) while 2C2-YTE was at 30 mg/kg. Calipermeasurements were used to calculate tumor volumes using the formula:

tumor volume=π÷6(length×width×width)

-   -   for tumors grown in mice. Antitumor effects are expressed as        percent delta tumor growth inhibition (TGI), which was        calculated as follows:

percent delta TGI=1−(dT÷dC)×100,

-   -   where dT=change in mean tumor volume in treatment group compared        to the value at staging, and dC=change in mean tumor volume in        control group compared to the value at staging.

4.10.2. Results.

2C2 at several different concentrations and 2C2-YTE at 10 mg/kgadministered twice per week displayed modest anti-tumor efficacy in thehuman HCT-116 colorectal xenograft model injected subcutaneously intofemale nude mice (FIG. 28). Maximal efficacy was noted at 43% dTGI for2C2 at 10 mg/kg. The anti-EGFR monoclonal antibody cetuximab had noefficacy at 10 mg/kg. The HCT-116 xenograft model contains a KRASmutation.

4.11. Subcutaneous Human LOVO CRC Xenograft Model Studies

4.11.1. Method.

Human LOVO colorectal carcinoma cells (ATCC No.CCL-229) were maintainedat 37° C. in a 5% CO₂ incubator in HAM'S F12K medium containing 4.5 g/Lglucose, L-glutamine, sodium pyruvate and 10% fetal bovine serum.Xenografts were established by subcutaneously injecting 5×10⁶ cells permouse into the right flanks of 4- to 6-week-old athymic nu/nu mice.Tumors were allowed to grow up to 200 mm³ before randomization forefficacy studies. 2C2, 2C2-YTE, cetuximab and control IgG1 monoclonalantibodies were administered intraperitoneally. 2C2 was administered at10 or 30 mg per kilogram body weight (mg/kg), 2C2-YTE and cetuximab wereadministered at 10 mg/kg and the control at 30 mg/kg. Calipermeasurements were used to calculate tumor volumes using the formula:

tumor volume=π÷6(length×width×width)

-   -   for tumors grown in mice. Antitumor effects are expressed as        percent delta tumor growth inhibition (TGI), which was        calculated as follows:

percent delta TGI=1−(dT÷dC)×100,

-   -   where dT=change in mean tumor volume in treatment group compared        to the value at staging, and dC=change in mean tumor volume in        control group compared to the value at staging.

4.11.2. Results

2C2 at 30 mg/kg administered twice per week achieved anti-tumor efficacyof 48% dTGI in the human LOVO colorectal xenograft model grownsubcutaneously in female nude mice (FIG. 29). 2C2, 2C2-YTE and cetuximaball at 10 mg/kg had comparable efficacy. The LOVO xenograft modelcontains a KRAS mutation.

4.12. Subcutaneous Human DU145 Prostate Carcinoma Xenograft ModelStudies

4.12.1. Method.

Human DU145 prostate carcinoma cells (ATCC No.HTB-81) were maintained at37° C. in a 5% CO₂ incubator in MEM medium containing Earle's salts,1-glutamine and 10% fetal bovine serum. Xenografts were established bysubcutaneously injecting 5×10⁶ cells per mouse (suspended in 50%matrigel) into the right flanks of 4- to 6-week-old athymic nu/nu mice.Tumors were allowed to grow up to 200 mm³ before randomization forefficacy studies. 2C2, MM and AMG monoclonal antibodies wereadministered intraperitoneally at 30 mg per kilogram body weight.Caliper measurements were used to calculate tumor volumes using theformula:

tumor volume=π÷6(length×width×width)

-   -   for tumors grown in mice. Antitumor effects are expressed as        percent delta tumor growth inhibition (TGI), which was        calculated as follows:

percent delta TGI=1−(dT÷dC)×100,

-   -   where dT=change in mean tumor volume in treatment group compared        to the value at staging, and dC=change in mean tumor volume in        control group compared to the value at staging.

4.12.2. Results

Using a human DU145 prostate cancer xenograft model grown subcutaneouslyin male nude mice 2C2 at 30 mg/kg administered twice per weekdemonstrated anti-tumor efficacy of 77% dTGI in this tumor model (FIG.30). The DU145 xenograft model contains a LKB-1 deletion. The anti-HER3monoclonal antibodies AMG and MM used at 30 mg/kg demonstratedanti-tumor efficacy but they were less effective than 2C2 at the samedose of 30 mg/kg.

4.13. Orthotopic Human BT-474 Breast Cancer Xenograft Model Studies

4.13.1. Method.

Human BT-474 breast cancer cells were maintained at 37° C. in a 5% CO₂incubator in RPMI 1640 medium containing 4.5 g/L glucose, L-glutamine,sodium pyruvate and 10% fetal bovine serum. Orthotopic xenografts wereestablished by injecting 1×10⁷ cells per mouse (suspended in 50%matrigel) into the mammary fat pad on the right flank of 4 to 6-week-oldathymic nu/nu mice. Estrogen pellets (0.36 mg) were placed under theskin of the left flank 1-2 days before cell injection. Tumors wereallowed to grow up to 200 mm³ before randomization for efficacy studies.2C2, 2C2-YTE, and or anti-HER2 antibodies known in the art: MM, AMG andtrastuzumab (trade name Herceptin®; e.g., U.S. Pat. No. 5,821,337) wereadministered intraperitoneally at 30 mg per kilogram body weight.Lapatinib was administered by oral gavaging at 100 mg per kilogram bodyweight. Caliper measurements were used to calculate tumor volumes usingthe formula:

tumor volume=π÷6(length×width×width)

-   -   for tumors grown in mice. Antitumor effects are expressed as        percent delta tumor growth inhibition (TGI), which was        calculated as follows:

percent delta TGI=1−(dT÷dC)×100,

-   -   where dT=change in mean tumor volume in treatment group compared        to the value at staging, and dC=change in mean tumor volume in        control group compared to the value at staging.

4.13.2. Results

Using a HER2-driven human breast cancer model, BT-474, injectedorthotopically into the mammary fat pad of female nude miceadministration of 2C2 at 30 mg/kg injected twice per week led to a 55%dTGI in BT-474 xenografts (FIG. 31A). BT-474 express HER2 at very highlevels of 3+ characterized by HercepTest. AMG and MM both administeredat 30 mg/kg did not show anti-tumor efficacy in this HER2-driven model.

Lapatinib is a small molecule drug inhibiting EGFR and HER2. SinceBT-474 tumors are driven by HER2, lapatinib was tested in this model andfound to cause tumor stasis in the BT-474 tumor model. The combinationtreatment of 30 mg/kg of 2C2 with 100 mg/kg of lapatinib resulted inimproved anti-tumor efficacy of lapatinib alone which was most clearlyvisible in a delay in regrowth of the tumors in the absence ofadditional treatments (FIG. 31B). The anti-tumor activity of 2C2-YTE wassimilar to that of 2C2. The anti-HER2 antibody trastuzumab was also wastested in this model and shown to be very active in this HER2-drivenxenograph model with a dTGI of 111.6%. There was little furtherenhancement in the activity of trastuzumab at 30 mg/kg by the additionof 30 mg/kg of 2C2 which showed a dTGI of 118.5% (FIG. 31C).

The ability of clone 16 (the parental clone from which 2C2 was derived)to modulate the pharmacodynamic markers pHER3 and pAKT was tested inBT-474 xenograft tumor extracts. Briefly, female athymic nude mice wereimplanted orthotopically with high HER2-expressing BT-474 breast cancercells. Animals were administered Clone 16 at 30 mg/kg twice within 48hours. Extracts were prepared 24 hours later for analysis of pHER3,pAKT, and total HER3 (tHER3). The results are normalized for PBS-treatedcontrol animals. There were three animals per treatment group. As shownin FIG. 32, Clone 16 inhibited phosphorylation of both HER3 and AKT by50.0% and 46.1%, respectively, compared to tumors from PBS-treated miceand no modulation of total HER3 was observed by Clone 16.

4.14. Orthotopic Human MCF-7 Breast Cancer Xenograft Model Studies

4.14.1. Method.

Human MCF-7 breast cancer cells were maintained at 37° C. in a 5% CO₂incubator in Optimem medium containing glutamax, 2.4/L sodiumbicarbonate, Hepes and 5% fetal bovine serum. Orthotopic xenografts wereestablished by injecting 5×10⁶ cells per mouse (suspended in 50%matrigel) into the mammary fat pad on the right flank of 4 to 6-week-oldathymic nu/nu mice. Estrogen pellets (0.36 mg) were placed under theskin of the left flank 1-2 days before cell injection. Tumors wereallowed to grow up to 200 mm³ before randomization for efficacy studies.2C2, 2C2-YTE and trastuzumab monoclonal antibodies were administeredintraperitoneally. 2C2 was administered at 10 or 30 mg per kilogram bodyweight (mg/kg) 2c2-YTE and trastuzumab at 10 mg/kg. Paclitaxel wasadministered intravenously at 10 mg per kilogram body weight. Calipermeasurements were used to calculate tumor volumes using the formula:

tumor volume=π÷6(length×width×width)

-   -   for tumors grown in mice. Antitumor effects are expressed as        percent delta tumor growth inhibition (TGI), which was        calculated as follows:

percent delta TGI=1−(dT÷dC)×100,

-   -   where dT=change in mean tumor volume in treatment group compared        to the value at staging, and dC=change in mean tumor volume in        control group compared to the value at staging.

4.14.2. Results

2C2 at either 10 mg/kg or 30 mg/kg showed modest anti-tumor efficacy of34% dTGI in a human MCF-7 breast cancer xenograft model injectedorthotopically into the mammary fat pad of female nude mice. 2C2-YTE at10 mg/kg had similar efficacy as 2C2 at the same concentration (FIG.33A). Trastuzumab did not demonstrate efficacy in this HER2 expressingmodel which indicated that HER2 is not sufficient to drive tumor growth.MCF-7 tumors expressed low levels of HER2 (1+) measured by HercepTest.

Paclitaxel showed clear anti-tumor efficacy in the MCF-7 orthotopicbreast cancer model when dosed at 10 mg/kg every second day for tendays. The addition of 10 mg/kg of 2C2 to the paclitaxel treatmentincreased the anti-tumor efficacy of paclitaxel alone at the end of thetreatment phase (FIG. 33B). The tumors regrew at the same rate as thepaclitaxel treated tumors after the treatment was stopped.

4.15. Orthotopic Human MDA-MB-361 Breast Cancer Xenograft Model Studies

4.15.1. Method.

Human MDA-MB-361 breast cancer cells were maintained at 37° C. in a 5%CO2 incubator in RPMI 1640 medium containing 4.5 g/L glucose,L-glutamine, sodium pyruvate and 10% fetal bovine serum. Orthotopicxenografts were established by injecting 5×10⁶ cells per mouse(suspended in 50% matrigel) into the mammary fat pad on the right flankof 4 to 6-week-old athymic nu/nu mice. Estrogen pellets (0.36 mg) wereplaced under the skin of the left flank 1-2 days before cell injection.Tumors were allowed to grow up to 230 mm³ before randomization forefficacy studies. 2C2-YTE, and/or anti-HER2 antibodies known in the art,in particular trastuzumab (trade name Herceptin®; e.g., U.S. Pat. No.5,821,337) and RhuMAb 2C4 (e.g., Patent Publication WO2001/00245)designated herein as trastuzumab and 2C4, respectively. Trastuzumab, and2C4 monoclonal antibodies were administered intraperitoneally at 30 mgper kilogram body weight (2C2-YTE) or at 10 mg per kilogram body weight(trastuzumab and 2C4). Lapatinib was administered by oral gavaging at100 mg per kilogram body weight. Caliper measurements were used tocalculate tumor volumes using the formula:

tumor volume=π÷6(length×width×width)

-   -   for tumors grown in mice. Antitumor effects are expressed as        percent delta tumor growth inhibition (TGI), which was        calculated as follows:

percent delta TGI=1−(dT÷dC)×100,

-   -   where dT=change in mean tumor volume in treatment group compared        to the value at staging, and dC=change in mean tumor volume in        control group compared to the value at staging.

4.15.2. Results

Using a HER2-driven human breast cancer model, MDA-MB-361 (Hercept test+2), injected orthotopically into the mammary fat pad of female nudemice, administration of 2C2-YTE at 30 mg/kg injected twice per week forfive doses led to a 70.1% dTGI in MDA-MB-361 xenografts (FIG. 34A-C).MDA-MB-361 cells express HER2 at medium levels of 2+ characterized byHercepTest and score positive by FISH analysis (fluorescent in situhybridization analysis). Trastuzumab and rhuMAb 2C4both administered at10 mg/kg and lapatinib at 100 mg/kg administered twice daily also showedanti-tumor efficacy in the MDA-MB-361 tumor model.

Since MDA-MB-361 tumors are driven by HER2, 2C2-YTE was combined withdrugs that target HER2 such as trastuzumab, rhuMAb 2C4 or lapatinib. Thecombination treatment of 30 mg/kg of 2C2-YTE with 10 mg/kg oftrastuzumab resulted in additive anti-tumor efficacy compared totrastuzumab alone. An additive effect was also visible in a delay inregrowth of the tumors in the absence of additional treatments (FIG.34A). The combination of 2C2-YTE with trastuzumab was better compared tocombinations of 2C2-YTE with either rhuMAb 2C4 (FIG. 34B) or lapatinib(FIG. 34C) in this model.

4.16. Transgenic Mice Expressing Human FcRn Receptor to Study Exposureof Antibodies with the YTE Modification.

4.16.1. Method.

Transgenic female SCID mice expressing the human FcRn receptor weregiven a single dose of 60 mg/kg of Clone 16-GL, 2C2 or 2C2-YTE via theintravenous route. Serum was collected from these mice at several timepoints after dosing by cardiac puncture and the blood was collected intoSST microtainer tubes. The tubes were vortexed gently for 10 seconds andkept at room temperature for 20 minutes to allow the serum to clot.Samples were centrifuged at 1000×g for 10 minutes, and the serum sampleswere carefully transferred into new tubes and stored at −80° C. Anindirect Enzyme-Linked Immunosorbent Assay (ELISA) format was used forthe quantitative determination of 2C2 in mouse serum. Standards, qualitycontrols, and mouse serum samples were incubated with goat anti-humanIgG antibodies which were immobilized on a 96-well microtiter plate.After incubation, unbound materials were removed by a wash step and 2C2was detected using a goat anti-human IgG with horseradish-peroxidaseconjugate. An acidic stopping solution was added and the degree ofenzymatic turnover of substrate was determined by measuring absorbanceat 450 nm. The absorbance measured was directly proportional to theconcentration of 2C2 or 2C2-YTE present in the mouse serum. A 2C2 or2C2-YTE standard curve for the assay was used to interpolate theconcentration of the serum samples.

4.16.2. Results.

2C2-YTE, which contains the YTE mutation on the 2C2 backbone, showedhigher exposure levels over time compared to 2C2 or Clone 16-GL (FIG.35). Fourteen days after the single dose of antibody to these mice theserum exposure level of 2C2-YTE was above 100 μg/ml while both 2C2 andClone 16-GL were below 1 μg/ml. This finding demonstrated that YTE couldextend the half-life of 2C2-YTE compared to its parental antibody 2C2.

4.17. MEK Inhibitor Induces HER3 Expression and in Combination withAnti-HER3 Antibody Shows Additive Anti-Tumor Efficacy.

KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene) and BRAF (v-rafmurine sarcoma viral oncogene B1) mutations lead to the constitutiveactivation of EGFR signaling through the oncogenic Ras/Raf/Mek/Erkpathway. Kras mutation is among the most-frequently occurring mutationevents in many solid tumors, especially colorectal (CRC, 30-40%) andlung cancers (LC, 20-25%). Braf mutation also occurs at relatively highfrequency in CRC (˜15%). Due to their ability to constitutively activatethe ERK pathway, mutant Kras and Braf have been shown to confer tumorresistance to RTK therapies, especially EGFR mAbs such as Cetuximab andPanitumumab. The effect of inhibiting mitogen-activated protein kinase(MEK) on the HER3 pathway in CRC and LC models was examined using theMEK inhibitor selumetinib (AstraZeneca, see for e.g., WO03/077914 andWO2007/076245) alone or in combination with 2C2 (or 2C2-YTE). A numberof CRC and LC models were examined including those harboring a mut-Kras(e.g. A549, LOVO) or mut-Braf (e.g., HT-29, Colo205) or a wild type RAS(e.g., HARA-B, KNS-62).

4.17.1. Methods.

Cell culture studies: cells were plated at 10⁵ per well in 24-wellplates and in medium containing 10% heat-inactivated FBS and allowed toreach a confluency of 80% or more prior to treatment. 2C2 (10 μg/mL) orcontrol antibody, MEK inhibitor selumetinib (1 or 10 μM) or acombination of 2C2 (10 μg/mL) and selumetinib (10 μM) were prepared incomplete medium. Treatments were applied following removal of platingmedium. After an incubation of 24 hours in 5% CO₂ at 37° C., cells werewashed once with ice-cold PBS and then lysed by adding 60 μL of 2×sodium dodecyl sulfate (SDS) sample buffer (Invitrogen). The sampleswere heated for 5 minutes and then chilled on ice for 2 minutes. Thesamples were analyzed by Western blotting essentially as described above(see Examples, section 2.4).

Xenograft studies: Human A549 NSCLC cells (ATCC No. CCL-185) whichcontain a mutation in codon 12 of the KRAS gene (were maintained at 37°C. in a 5% CO₂ incubator in HAM′S F12K medium containing 4.5 g/Lglucose, L-glutamine, sodium pyruvate and 10% fetal bovine serum.Xenografts were established by subcutaneously injecting 5×10⁶ cells permouse (suspended in 50% matrigel) into the right flanks of 4- to6-week-old athymic nu/nu mice. Human HT-29 colorectal carcinoma cells(ATCC No. HTB-38) were maintained at 37° C. in a 5% CO₂ incubator inRPMI 1640 medium containing 4.5 g/L glucose, L-glutamine, sodiumpyruvate and 10% fetal bovine serum. Xenografts were established bysubcutaneously injecting 5×10⁶ cells per mouse into the right flanks of4- to 6-week-old athymic nu/nu mice. Human LOVO colorectal carcinomacells (ATCC No. CCL-229) were maintained at 37° C. in a 5% CO₂ incubatorin HAM′S F12K medium containing 4.5 g/L glucose, L-glutamine, sodiumpyruvate and 10% fetal bovine serum. Xenografts were established bysubcutaneously injecting 5×10⁶ cells per mouse into the right flanks of4- to 6-week-old athymic nu/nu mice. For all three tumor models, tumorswere allowed to grow up to 200 mm³ before randomization for efficacystudies. 2C2-YTE or control IgG1 were administered intraperitoneally.selumetinib was administered orally. For the combination studies 2C2-YTEand selumetinib were administered at 30 mg/kg or 75 mg/kg, respectively.Caliper measurements were used to calculate tumor volumes using theformula:

tumor volume=π÷6(length×width×width)

-   -   for tumors grown in mice. Antitumor effects are expressed as        percent delta tumor growth inhibition (TGI), which was        calculated as follows:

percent delta TGI=1−(dT÷dC)×100,\

-   -   where dT=change in mean tumor volume in treatment group compared        to the value at staging, and dC=change in mean tumor volume in        control group compared to the value at staging.

Preparation of lysates from frozen tumors: Mice were humanely euthanizedby CO₂ asphyxiation in accordance with our in vivo protocol and tumorswere excised and transferred to Lysing Matrix A tubes. RIPA lysis buffer(500 μL) containing protease inhibitor cocktail and phosphataseinhibitor cocktail set I and II was added, the samples were thenhomogenized using a Fast Prep machine. Samples were chilled on ice for30 minutes and underwent an additional homogenization cycle beforeclarification by centrifugation at 14,000 rpm for 10 minutes at 4° C.Clarified lysates were transferred to fresh 1.5 mL tubes and proteincontent was measured. Lysates were then stored at −80° C. untilanalysis. The samples were analyzed by Western blotting essentially asdescribed above (see Examples, section 2.4).

4.17.2. Results.

As shown in FIG. 36, both total and pHER3 protein levels increasedfollowing treatment with the MEK inhibitor selumetinib in HT-29colorectal cancer cells grown in culture which express mutant BRAF andin LOVO cells which express a mutant KRAS (FIG. 36, left and middleblots respectively). An increase of HER3 was also observed in Colo205cells which express mut-BRAF and in DLD-1 and HCT cells, which expressmutant KRAS (FIG. 36, right blot, and data not shown), followingselumetinib treatment. The increases occurred at both the 1 μM and 10 μMdoses of selumetinib. Activity of selumetinib was confirmed by reductionin pERK in all cell lines at both 1 μM and 10 μM doses. Inhibition ofMEK results in an inhibition of ERK phosphorylation. The anti-HER3antibody, 2C2, inhibited both total and pHER3 in HT-29 and LOVO cells.2C2 also lowered HER3 in Colo205 and DLD-1 cells. In addition,co-treatment of 2C2 with selumetinib blocked the induction of total HER3and pHER3 by selumetinib in HT-29, LOVO and DLD-1 cells (FIG. 36, anddata not shown). No detectable HER3 or pHER3 could be observed in SW480colorectal cancer cells, which express mutant KRAS, in either untreatedor selumetinib -treated cells.

As shown in FIG. 37A, the combination treatment of 30 mg/kg of 2C2-YTEwith 75 mg/kg of selumetinib resulted in additive anti-tumor efficacy inA549 NSCLC xenografts compared to selumetinib alone. An additive effectwas also visible in a delay in regrowth of the tumors in the absence ofadditional treatments (top panel). Western blot analysis of tumorlysates from mice treated with the combination of 30 mg/kg of 2C2-YTEwith 75 mg/kg of selumetinib over a 4 day period showed thatphospho-HER3 and phospho-ERK were completely inhibited. Both markersserve as pharmacodynamic read-outs for the action of 2C2-YTE andselumetinib. Similar findings were made with the CRC xenograft modelsHT-29 (FIG. 37 B, upper and lower panel) and LoVo (FIG. 37C, upper andlower panel). In addition, phospho-AKT was found to be reduced in HT-29tumor lysates treated with the combination of 30 mg/kg of 2C2-YTE with75 mg/kg of selumetinib compared to single treatments (FIG. 37B, lowerpanel). Treatment with selumetinib alone at 75 mg/kg lead to an increasein phospho-HER3 in the LoVo tumor extracts which was prevented in tumorstreated with the combination of 2C2-YTE and selumetinib (FIG. 37C, lowerpanel). Similar results were seen in HARA-B (data not shown).

In cell culture the levels of HER3 protein were seen to increase inresponse to MEK inhibitor across most models examined, indicating thatthe HER3 pathway may play a role in resistance to MEK inhibitors. In anumber of orthotopic CRC and LC xenograft model studies the combinationof 2C2-YTE and selumetinib was seen to increase the anti-tumor efficacyof either agent alone. These data support the use of 2C2 in combinationwith a MEK inhibitor like selumetinib to enhance anti-tumor activity andprevent resistance.

4.18. Toxicology Studies in Cynomolgus Monkey

4.18.1. Method

Twenty Male cynomolgus monkeys (Macaca fascicularis) were assigned tofour groups (5 animals per group) and a total of five doses of vehiclecontrol or 2C2-YTE at 10, 30 or 120 mg/kg were administered. Animalswere dosed once weekly via 5-minute IV infusion at a dose volume of 5mL/kg. Three animals per group were necropsied on Day 32 (three daysafter the final dose administration on Day 29 of the dosing phase) andtwo animals per group were necropsied on Day 43 of the recovery phase(forty-five days after the final dose administration on Day 29 of thedosing phase). Assessment of toxicity was based on a number of factorsincluding mortality, clinical observations, body weights, dose siteirritation scoring, clinical and anatomic pathology evaluations.

Cynomolgus monkey plasma samples were isolated and analyzed for solubleHER3 (sHER3) levels using an anti-HER3 sandwich format with anelectrochemiluminescence (ECL) detection system for quantitation of freesHER3. Meso Scale Discovery (MSD) bare 96-well plates (MSD, catalognumber L15XA-6/L11XA-6) were coated with 0.5 ug/ml of 2C2-YTE overnightat 2 to 8° C. and subsequently blocked with MSD Blocker A (MSD, catalognumber R93BA-1). Reference Standard and Quality controls (QC), andcynomolgus monkey plasma undiluted test samples were added to blockedplates for 1 hour at room temperature. Biotinylated anti-hErbB3/HER3antibody (R&D Systems, catalog number BAM348) followed by addition ofSulfo-TAG (MSD, catalog number R32AD-1) resulted in light emission whenelectrochemically stimulated. The ECL signal was captured and recordedon a MSD Sector Imager 2400. The amount of light generated directlycorrelated with the amount of sHER3 in the cynomolgus monkey plasmasamples. The raw data (ECL counts) were exported into SOFTmax® PRO. Thestandard curve for recombinant human HER3 standards was fitted using a5-parameter fit program. Cynomolgus monkey plasma HER3 concentrationswere calculated based on the standard curve using the statisticalfunction of SOFTmax PRO.

In addition, skin biopsy samples were collected for bioanalysis.Briefly, matched 10 mm circles are drawn on the skin on the animal and˜100 μL of PBS or HRG at 0.1 mg/mL was injected intradermally into thecenter of each circle. Approximately 20 minutes later a skin sample wascollected from each injection site and flash frozen. Alternatively,matched biopsy samples are collected (without prior intradermalinjection) from each and incubated for approximately 30 min at roomtemperature in culture media with or without 100 μg/mL HRG followed bytwo washes with ice-cold PBS. The washed sample is then flash-frozen.The tissues were then homogenized in Lysing Matrix A tubes (MPBiomedicals) containing RIPA lysis buffer and protease inhibitorcocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail set I and II(EMD-Millipore) using a Fast Prep machine (MP Biomedicals). Samples werethen subjected to a freeze-thaw cycle and an additional homogenizationcycle before clarification by centrifugation at 14,000 rpm for 5 minutesat 4° C. Clarified lysates were transferred to fresh 1.5 mL tubes andprotein content was measured. The levels of total HER3 and pHER3 aredetermined using a sandwich ELISA assay.

4.18.2. Results

A non-GLP, 1-month, repeat-dose toxicity study of 2C2-YTE with asix-week recovery phase was performed in cynomolgus monkeys to evaluatethe toxicity and activity of 2C2-YTE, when administered once weekly viaan IV infusion to cynomolgus monkeys for at least 1-month (5 totaldoses) and to assess the reversibility, persistence, or delayedoccurrence of any effects after a 6-week recovery period. No adverseeffects were noted following once weekly IV administration (5 minuteinfusion) of up to 120 mg/kg/dose of 2C2-YTE, for 5 weeks (5 totaldoses), in male cynomolgus monkeys.

The ability of 2C2-YTE to block HRG-induced pHER3 in the skin ofcynomolgus monkeys was confirmed by in vivo and ex vivo evaluations.Complete suppression of circulating soluble HER3 was observed in allanimals receiving intravenous 2C2-YTE. Ex-vivo stimulation of skinbiopsies with HRG resulted in an increase in the pHER3:tHER3 ratio,demonstrating that HER3 present in the skin of cynomolgus monkeys can beactivated by HRG, the predominant ligand for HER3. Complete suppressionof HRG-induced pHER3 was achieved in all 2C2-YTE treated groups at theend of the dosing phase (data not shown). Thus, 2C2-YTE blocked in vivoand ex vivo HRG-induced HER3 phosphorylation in cynomolgus monkey skinbiopsy samples.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In addition, U.S. Provisional Application Nos. 61/563,092filed Nov. 23, 2011; 61/656,670 filed Jun. 7, 2012; and 61/722,558 filedNov. 5, 2012, are incorporated by reference in their entirety for allpurposes.

The preceding description of the specific aspects will so fully revealthe general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific aspects, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed aspects, based on the teaching and guidance presented herein.It is to be understood that the phraseology or terminology herein is forthe purpose of description and not of limitation, such that theterminology or phraseology of the present specification is to beinterpreted by the skilled artisan in light of the teachings andguidance.

1.-119. (canceled)
 120. A method of treating cancer in a subject,comprising administering to the subject a therapeutically effectiveamount of an isolated antibody or an antigen-binding fragment thereof,wherein the antibody or an antigen-binding fragment thereof specificallybinds to HER3 and comprises an antibody variable light chain region (VL)and an antibody variable heavy chain region (VH), wherein the VLcomprises the amino acid sequence: [FW1]SGSLSNIGLNYVS (SEQ ID NO: 19)[FW2]RNNQRPS (SEQ ID NO: 21) [FW3]AAWDDSPPGEA (SEQ ID NO: 23) [FW4]

wherein [FW1], [FW2], [FW3] and [FW4] represent VL framework regions,and wherein the VH comprises the amino acid sequence: [FW5]YYYMQ(SEQ ID NO: 31) [FW6]YIGSSGGVTNYADSVKG (SEQ ID NO: 32) [FW7]VGLGDAFDI(SEQ ID NO: 35) [FW8]

wherein [FW5], [FW6], [FW7] and [FW8] represent VH framework regions.121. The method of claim 120, wherein the cancer is a carcinoma. 122.The method of claim 120, wherein the cancer is selected from the groupconsisting of colorectal cancer, lung cancer, gastric cancer, breastcancer, head and neck cancer, prostate cancer, pancreatic cancer,thyroid cancer, melanoma, esophageal cancer, ovarian cancer, kidneycancer, and bladder cancer.
 123. The method of claim 122, wherein thecancer is a squamous cell carcinoma of the head and neck.
 124. Themethod of claim 122, wherein the cancer comprises cells comprising aKRAS mutation.
 125. The method of claim 120, wherein the method furthercomprises administering to the subject a therapeutically effectiveamount of a second agent, wherein the second agent is an anti-canceragent other than the antibody or antigen-binding fragment thereof. 126.The method of claim 125, wherein the second agent is an EGFR inhibitor,a HER2 inhibitor, a HER3 inhibitor, or a MEK inhibitor.
 127. The methodof claim 125, wherein the second agent is cetuximab, panitumumab,matuzumab, nimotuzumab, MM-151, or Sym004.
 128. The method of claim 125,wherein the second agent is trastuzumab, trastuzumab emtansine, orpertuzumab.
 129. The method of claim 125, wherein the second agent is akinase inhibitor.
 130. The method of claim 125, wherein the second agentis gefitinib, canertinib, lapatinib, erlotinib, afatinib, neratinib,selumetinib, WX-554, selumetinib, trametinib, refametinib, E-6201, orMEK-162.
 131. The method of claim 120, wherein the cancer ischaracterized as expressing heregulin.
 132. The method of claim 120,wherein the VL comprises an amino acid sequence at least 80% identicalto the amino acid sequence of SEQ ID NO: 3, and wherein the VH comprisesan amino acid sequence at least 80% identical to the amino acid sequenceof SEQ ID NO:
 2. 133. The method of claim 120, wherein the VL comprisesan amino acid sequence at least 90% identical to the amino acid sequenceof SEQ ID NO: 3, and wherein the VH comprises an amino acid sequence atleast 90% identical to the amino acid sequence of SEQ ID NO:
 2. 134. Themethod of claim 120, wherein the VL comprises an amino acid sequence atleast 95% identical to the amino acid sequence of SEQ ID NO: 3, andwherein the VH comprises an amino acid sequence at least 95% identicalto the amino acid sequence of SEQ ID NO:
 2. 135. The method of claim120, wherein FW1 comprises SEQ ID NO: 40 or 44, FW2 comprises SEQ ID NO:41, FW3 comprises SEQ ID NO: 42, FW4 comprises SEQ ID NO: 43, FW5comprises SEQ ID NO: 36, FW6 comprises SEQ ID NO: 37, FW7 comprises SEQID NO: 38 and FW8 comprises SEQ ID NO:
 39. 136. The method of claim 120,wherein the antibody or antigen-binding fragment thereof comprises a VLcomprising SEQ ID NO: 3 and a VH comprising SEQ ID NO:
 2. 137. Themethod of claim 120, wherein the antibody or antigen-binding fragmentthereof comprises a heavy chain constant region.
 138. The method ofclaim 137, wherein the heavy chain constant region is a human IgGconstant region.
 139. The method of claim 138, wherein the human IgGconstant region is an IgG1 constant region.
 140. The method of claim120, wherein the antibody or antigen-binding fragment thereof comprisesa light chain constant region selected from the group consisting of ahuman kappa constant region and a human lambda constant region.
 141. Themethod of claim 139, wherein the antibody or antigen-binding fragmentthereof comprises a light chain constant region selected from the groupconsisting of a human kappa constant region and a human lambda constantregion.
 142. The method of claim 141, wherein the antibody orantigen-binding fragment thereof comprises a human lambda constantregion.
 143. The method of claim 138, (i) wherein the human IgG constantregion comprises amino acid substitutions relative to a wild-type humanIgG constant domain at positions 252, 254, and 256, wherein thenumbering is according to the EU index as set forth in Kabat, andwherein (a) the amino acid at position 252 (Methionine) is substitutedwith Tyrosine (Y), (b) the amino acid at position 254 (Serine) issubstituted with Threonine (T), and (c) the amino acid at position 256(Threonine) is substituted with Glutamic acid (E).
 144. The method ofclaim 120, wherein the antibody is a human antibody, a humanizedantibody, a chimeric antibody, a recombinant antibody, a multispecificantibody, or an antigen-binding fragment thereof; wherein theantigen-binding fragment is an Fv, Fab, F(ab′)2, Fab′, dsFv, scFv, orsc(Fv)2; or wherein the antibody or antigen-binding fragment thereof isconjugated to at least one heterologous agent.
 145. The method of claim120, wherein the method comprises administering a composition comprisingthe antibody or antigen-binding fragment thereof and a pharmaceuticallyacceptable carrier.
 146. The method of claim 145, wherein thecomposition is refrigerator stable, wherein the antibody orantigen-binding fragment thereof is at a concentration of 25-100 mg/mL,and wherein the composition further comprises 25 mM histidine/histidineHCl, 205 mM sucrose, and 0.02% polysorbate 80 at pH 6.0.
 147. The methodof claim 120, wherein the antibody or antigen-binding fragment thereofis a monoclonal antibody.
 148. The method of claim 120, wherein theantibody or antigen-binding fragment thereof comprises (i) an antibodylight chain comprising an antibody VL comprising SEQ ID NO: 3 and ahuman lambda light chain constant region, and (ii) an antibody heavychain comprising an antibody VH comprising SEQ ID NO: 2 and a human IgG1heavy chain constant region, wherein the human IgG1 constant regioncomprises amino acid substitutions relative to a wild-type human IgG1constant domain at positions 252, 254, and 256, wherein the numbering isaccording to the EU index as set forth in Kabat, and wherein (a) theamino acid at position 252 (Methionine) is substituted with Tyrosine(Y), (b) the amino acid at position 254 (Serine) is substituted withThreonine (T), and (c) the amino acid at position 256 (Threonine) issubstituted with Glutamic acid (E).
 149. The method of claim 144,wherein the antibody is a human antibody.
 150. The method of claim 148,wherein the cancer is head and neck cancer.
 151. The method of claim148, wherein the cancer is a squamous cell carcinoma of the head andneck.
 152. The method of claim 148, wherein the cancer is a squamouscell carcinoma of the head and neck, and wherein the method furthercomprises administering to the subject a therapeutically effectiveamount of cetuximab.
 153. The method of claim 148, wherein the cancer iscolorectal cancer.
 154. The method of claim 148, wherein the cancer isnon-small cell lung cancer.
 155. The method of claim 148, wherein thecancer is thyroid cancer.
 156. The method of claim 148, wherein thecancer is breast cancer.
 157. The method of claim 148, wherein thecancer is a HER2-amplified cancer.
 158. The method of claim 148, whereinthe cancer is melanoma.
 159. The method of claim 148, wherein the canceris gastric cancer.
 160. The method of claim 148, wherein the cancer isprostate cancer.